Nucleic acids involved in the responder phenotype and applications thereof

ABSTRACT

The present invention relates to nucleic acid molecules encoding expression products involved in the Responder function, which contributes to the phenomenon of transmission ratio distortion. The present invention also relates to regulatory regions of the genes corresponding to the nucleic acid molecules. The present invention further relates to recombinant DNA molecules and vectors comprising the nucleic acid molecules and/or regulatory regions as well as to host cells transformed therewith. Additionally, the present invention relates to transgenic animals, comprising the nucleic acid molecules, recombinant DNA molecules or vectors stably integrated into their genome. The various embodiments of the invention have a significant impact on breeding strategies by allowing for the specific selection of genetic traits and in particular of sex. Further, the present invention finds applications in the development of contraceptive.

The present application is a U.S. National Stage Application of PCT/EP 98/07395, filed Nov. 18, 1998. This application also claims the benefit under 35 U.S.C. §119 of foreign application nos. EP 97 12 0190.0, filed Nov. 18, 1997 and EP 98 10 3596.7 (Mar. 2, 1998).

The present invention relates to nucleic acid molecules encoding expression products involved in the Responder function, which contributes to the phenomenon of transmission ratio distortion. The present invention also relates to regulatory regions of the genes corresponding to said nucleic acid molecules. The present invention further relates to recombinant DNA molecules and vectors comprising said nucleic acid molecules and/or regulatory regions as well as to host cells transformed therewith. Additionally, the present invention relates to transgenic animals, comprising said nucleic acid molecules, recombinant DNA molecules or vectors stably integrated into their genome. The various embodiments of the invention have a significant impact on breeding strategies by allowing for the specific selection of genetic traits and in particular of sex. Further, the present invention finds applications in the development of contraceptive.

The mouse T/t-complex, a region of approximately 12 cM genetic distance on the proximal part of chromosome 17, contains several loci acting in concert to produce a phenomenon called transmission ratio distortion (TRD). The latter designation indicates the fact that the so-called t-haplotype form of this chromosomal region has a selective advantage over the wild type form in that it is transmitted to the offspring at non-mendelian ratios of up to 99%. This transmission at non-mendelian ratio is achieved by the concerted action of mainly four loci, the so-called Distorters Tcd-1 (D1), Tcd-2 (D2) and Tcd-3 (D3), and the Responder Tcr (R^(t))(Lyon 1984). Two more Distorters have been postulated by other authors (Silver and Remis 1987).

According to Lyon's model (Lyon 1986) which formally explains the genetic interactions of these loci, the Distorters D1, D2 and D3 act strongly and harmfully on the wild type allele of the Responder and weakly on the t form of the Responder (R^(t)), leading to distortion in favor of R^(t). R^(t) might protect sperm carrying it from this harmful action of the Distorters. The Distorters act in trans while the Responder acts in cis. This means that the chromosome which contains R^(t) is transmitted at non-mendelian ratio to the offspring. If D2 or all the Distorters are present, the chromosome containing R^(t) is transmitted at a frequency of more than 50% up to 99% to the offspring. If no Distorter or only D1 or D3 are present, however, the chromosome containing R^(t) is transmitted at less than 50% to the offspring (“low” phenotype). The Distorters are only transmitted at ratios over 50% if they are located on the same chromosome as is R^(t). The cis-action of R^(t) suggests that R^(t) may be expressed at a stage of spermiogenesis when spermatids are no longer connected in a syncytium (Willison and Ashworth 1987). This would ensure that the product of R^(t) would be restricted to the spermatozoon containing the t-haplotype form of the R locus. It is expected that expression in elongating spermatids or mature spermatozoa is compatible with this requirement. The trans-acting and cis-acting properties of the Distorters and the Responder, respectively, have been demonstrated by the transmission ratio properties of so-called partial t-haplotypes which carry only a subset of the above named loci (FIG. 1).

Genetic mapping of molecular markers on partial t-haplotypes allowed a more or less precise localization of D1, D2, D3 and R^(t) to subregions of the T/t-complex and relative to these molecular markers (Lyon 1984; Fox et al. 1985; Herrmann et al. 1986; Silver and Remis 1987; Bullard et al. 1992). Only one locus, R^(t) could be mapped fairly precisely to a region of appr. 200 kb, the so-called T66B region (Fox et al. 1985; Schimenti et al. 1987; Nadeau et al. 1989; Rosen et al. 1990; Bullard et al. 1992). The T66B region represents a chromosomal piece of the t-haplotype identified by a t-specific restriction fragment length polymorphism detected with the probe Tu66 (Fox et al. 1985). The T66B region is not present in the partial t-haplotypes t^(h44) and t^(h51), but is present in the partial t-haplotypes t^(low), t^(h2), t^(h49), t⁶, and in the complete t-haplotypes, e.g. t^(w5) or t^(w12) (FIG. 1). Another partial t-haplotype, t^(w71Jr1) (abbr. t^(Jr1)) contains T66A and a part of T66B. The chromosomes t^(h44), t^(h51)and t^(Jr1) do not contain the R^(t) function, whereas the other partial and complete t-haplotypes named above do. The t-haplotypes containing R^(t) function must have the t-form of R, whereas the haplotypes t^(h44), t^(h51) and t^(Jr1) are expected to have the wild type form. The genomic region T66B has been cloned molecularly and analyzed. A partial restriction map covering appr. 145 kb of it has been published (Schimenti et al. 1987; Rosen et al. 1990; Bullard et al. 1992).

An extensive and careful search of this region for genes expressed during spermatogenesis has yielded a single gene, T66B-a or Tcp-10b^(t) (Schimenti et al. 1988). Further mapping studies localized “sequences responsible for differential responder activity” to an interval of 40 kb at the telomeric end of the T66B region which includes Tcp-10b^(t) (Bullard et al. 1992). No other transcription unit could be identified by these authors in the T66B region within the last 10 years. Tcp-10b^(t) has been claimed to represent the candidate for R^(t), but a careful analysis showed that it does not encode Responder properties (Schimenti et al. 1988; Cebra-Thomas et al. 1991; Bullard and Schimenti 1990; Ewulonu et al. 1996).

The combined teachings of the prior art thus did not provide any clue how the genetic elements responsible for the Responder phenomenon might be identified. More importantly, the analyses referred to above questioned the prior art discussions that the Responder is a transcription unit. Accordingly, they taught away from the possibility that a transcription unit encoding the Responder might be located in the T66B region. The technical problem underlying the present invention was, accordingly, to overcome these long standing prior art difficulties and provide a genetic entity encoding the Responder function.

The solution to said technical problem is achieved by providing the embodiments characterized in the claims.

Accordingly, the present invention relates to a nucleic acid molecule comprising a transcription unit encoding in its 5′ portion a kinase having a homology to the MARK2 kinase (Drewes et al., 1997) as well as to other kinases whereas the 3′ portion of the nucleotide sequence has a high homology to the rsk3 kinase (Zhao et al., 1995) as well as to expression products thereof. The term “homology” as used in accordance with the present invention relates to more than 25% and preferably about 38% identity on the amino acid level. Thus, in accordance with the present invention, 38% identity was found in a region of 291 amino acids between MARK2 and the protein encoded by the nucleic acid molecule shown in FIGS. 4a and b or 9 a and b. Preferably, the kinase gene encoded by the 5′portion lacks its 3′ end which is preferably an untranslated region whereas the kinase gene encoded by the 3′ portion lacks the 5′ end and is preferably not translated.

Preferably or alternatively, the present invention relates to a nucleic acid molecule encoding an expression product involved in the Responder phenotype, which contributes to the phenomenon of transmission ratio distortion, selected from the group consisting of

(a) a nucleic acid molecule comprising the nucleic acid molecule as shown in FIGS. 4a and b or 9 a and b, 7 a and b, 7 c, d, and e, 7 f, g, and h, 7 i, j, and k, 7 l or a fragment thereof;

(b) a nucleic acid molecule being an allelic variant or a homologue of the nucleic acid sequence of (a);

(c) a nucleic acid molecule hybridizing to a nucleic acid molecule complementary to a nucleic acid molecule of (a) or (b); and

(d) a nucleic acid molecule which is related to the nucleic acid molecule of (a), (b) or (c) by the degeneration of the genetic code.

The term “Responder” or “R” as used in accordance with the present invention relates to mutant as well as wild type forms of the Responder locus.

The term “involved in the Responder phenotype”, in accordance with the present invention relates to the fact that transcripts of all genes displayed on FIGS. 4a and b or 9 a and b, 7 a and b, 7 c, d, and e, 7 i, j, and k and the antisense transcript of 7 f, g, and h are detected in testis carrying complete t-haplotypes, whereas mapping of the genes displayed on FIGS. 4a and b or 9 a and b and 7 a and b to the t-Responder region suggests that gene 4 a and b or 9 a and b and/or 7 a and b is (are) the one(s) encoding t-Responder activity. In accordance with the further biological data described in this specification, in particular the data relating to the transgenic animals, it is proposed that pursuant to this invention, the gene displayed in FIGS. 4a and b or 9 a and b encodes t-Responder activity. The overall data suggest that several genes of the Responder (T66Bk) gene family may act in parallel within t-haplotype carrying spermatids and/or spermatozoa and are thus presumed to be involved in the Responder phenotype, whereby it is envisaged that t-Responder products may antagonize the negative effect of t-Distorter genes and antisense transcripts derived from Responder genes may reduce the activity of Responder genes encoding products with t-Responder as well as wild type or nearly wild-type Responder activity. The latter products may permit the negative action of t-Distorter genes. It is, furthermore, envisaged in accordance with the present invention that alternative translation products from one mRNA-transcript may also be involved in the Responder phenotype (see, e.g., FIGS. 13a and b).

Specifically the cDNA sequence of T66Bk shown in FIGS. 4a and b or 9 a and b contains the MARK kinase and the rsk3 kinase homology regions. The cDNA sequence of T66Bk-2 shown in FIGS. 7a and b contains only the MARK kinase homology region. The cDNA sequence of T66k-8 shown in FIGS. 7c, d, and e contains the complete sequence of T66Bk-2 except for a single base deleted between nucleotide position 1508 and 1509 resulting in a frame shift. The cDNA sequence of T66k-7as shown in FIGS. 7f, g, and h corresponds to an antisense transcript of a T66Bk family member. The cDNA sequence of T66k-20 shown in FIGS. 7i, j, and k shows a strong similarity to the above members of the T66Bk gene family.

The term “fragment” as used in connection with the nucleic acid molecule of the invention relates to a fragment that retains the Responder function. Preferably, said fragment comprises the portion of the nucleic acid molecule that has a homology to the MARK kinase referred to above or a part thereof.

As has been indicated above, in one embodiment of the nucleic acid molecule of the invention said expression product is an antisense RNA.

The term “an allelic variant or a homologue” comprises forms of the wild type or t-allele of the Responder “gene” which have been manipulated in vitro in order to achieve an optimal transmission ratio distortion effect and/or to adapt it to the specific requirements of the breeding scheme employed, thus improving the selectability of genetic traits. A number of standard manipulations known in the field are taken into consideration, such as those resulting in the exchange of phosphorylation sites (Ser, Thr, Tyr) on the Responder (poly)peptide for acidic or neutral (Ala) amino acid residues, mutagenesis of the active center, overexpression or knock out mutagenesis of said gene, construction of hypomorphic (poly)peptides by mutagenesis of ATP and/or GTP binding site(s), deletion of phosphorylation sites on said (poly)peptide, deletion of binding sites for other (poly)peptides involved in the Responder/Distorter signaling cascade, synthesis of antisense RNA, N-terminal or C-terminal truncations, introduction of frame shifts which alter part of the amino acid sequence of the protein, etc., resulting either in null, hypomorphic, constitutively active, antimorphic or dominant negative alleles. It is also envisaged that a distortion of the transmission ratio can be achieved with several, if not all, manipulated forms of the Responder gene suggested above. Thus, a manipulated Responder allele affecting the transmission ratio most effectively will have to be identified empirically by employing activity assays in cell culture systems and by employing transgenic animal systems.

It is also envisaged that one or several members of the T66Bk kinase gene family might function as Distorter(s), provided it is (they are) expressed during the diploid or early haploid phase of spermatogenesis allowing distribution of the gene products to all spermatozoa, or can be altered in vitro such as to function as Distorters. The latter may be achieved by in vitro manipulations such as those resulting in the exchange of phosphorylation sites (Ser, Thr, Tyr) on said Responder (poly)peptide for acidic or neutral (Ala) amino acid residues, N- or C-terminal truncation, frame shift, deletion of phosphorylation sites, deletion of binding sites for other (poly)peptides, mutagenesis of the active center, or overexpression of said gene or of antisense transcripts, resulting in constitutively active, hypomorphic, antimorphic or dominant negative gene products and expression of said gene products during the diploid or early haploid phase of spermatogenesis allowing distribution of the gene products to all spermatozoa, e.g. under the control of the Pgk2 promoter. These manipulations are envisaged to have a negative effect on sperm motility and/or fertilization capability. This negative effect may then be balanced by Responder constructs having the opposite effect. The latter could be restricted to those spermatozoa carrying the construct by expressing it under the control of the Responder gene promoter. It is envisaged that both types of spermatozoa would be negatively affected by the Distorter construct expressed in the diploid phase of spermatogenesis, whereas the sperm carrying, in addition, the Responder construct expressed in spermiogenesis would be partially or completely protected by the (poly)peptide expressed in it, and would thus gain an advantage in sperm motility and/or fertilization capability over the other sperm. This would lead to a transmission ratio distortion in favor of the “protected” spermatozoa. Preferably the Distorter construct expressed in both types of spermatozoa would encode a hypermorphic or constitutively active (poly)peptide, whereas the Responder construct expressed only in those spermatozoa carrying it should encode a hypomorphic, antimorphic or dominant negative (poly)peptide. Both constructs could be integrated on the same or on different chromosomes. Preferably both constructs would be integrated together on the X- or the Y-chromosome, resulting in the preferential or exclusive transmission of the X- or Y-chromosome and thus the preferential or exclusive fathering of female or male offspring, respectively.

The term “hybridizing” as used in connection with the present invention relates to stringent or nonstringent hybridization conditions. Preferably, it relates to stringent conditions. Said hybridization conditions may be established according to conventional protocols described, for example, in Sambrook, “Molecular Cloning, A Laboratory Manual”, Cold Spring Harbor Laboratory (1989) N.Y., Ausubel, “Current Protocols in Molecular Biology”, Green Publishing Associates and Wiley Interscience, N.Y. (1989), or Hames et al., (eds) “Nucleic acid hybridisation, a practical approach” IRL Press Oxford, England, (1985).

Stringent hybridization conditions are, for example, hybridization in 6×SSC, 5×Denhardt's reagent, 0,5% SDS, and 100 μg/ml denatured DNA at 65° C. and washing in 0,1×SSC, 0,1% SDS at 65° C.

In accordance with the present invention and in contrast to the teachings of the prior art, it was surprisingly found that nucleic acid sequences responsible for the Responder phenotype are comprised at the centromere-close part of the T66 B region. It conforms with several criteria that would be expected for the Responder function:

a) it is located in the T66B region;

b) it is expressed in testis; and

c) it is expressed during spermatogenesis.

In accordance with the present invention, it is further envisaged that additional expression products may contribute to Responder function as has been indicated above which are not necessarily located in the B-region.

As has been indicated above, one of the transcription units (namely T66Bk) contributing to the Responder (R) phenotype apparently arises from two truncated genes. One of said genes has a high homology to the rsk3 gene, the second one has an homology to the MARK kinase recently identified (Drewes et al., 1997). Another transcription unit envisaged to contribute to the R phenotype, T66Bk-2, also has a homology to the MARK kinase, but lacks homology to the rsk3 gene as indicated above. The identification of the genetic basis underlying the R phenotype allows a number of genetic manipulations, in particular in connection with breeding schemes, to be conveniently carried out in the future. Such schemes will be addressed in more detail herein below.

In accordance with the present invention, it is envisaged that the expression products encoded by the nucleic acid sequences of the invention may contribute to the Responder phenotype in several different ways. Thus, in one embodiment one of the above indicated expression products are themselves sufficient to distort the transmission ratio. In another embodiment all of said expression products or combinations of them have to be provided in order to distort the transmission ratio, with certain combinations being more effective than others. In yet another embodiment of the present invention said expression products may work in an additive or synergistic manner. In a still further embodiment it is envisaged that antisense transcripts derived from one or several genes of the T66Bk gene family may contribute to the t-Responder function resulting in a lower level or abolishment of mRNA of one or several T66Bk genes and thus a lower level or abolishment of the corresponding (poly)peptides translated from said mRNA molecules. An example of such an antisense transcript is shown in FIGS. 7f, g, and h. Furthermore, it is suggested that the specifically identified nucleic acid sequences coding for expression products involved in the R phenotype may not be the only ones responsible for the Responder phenotype. Thus, it is envisaged that further nucleic acids encoding expression products that act in concert with the ones discussed above and that may contribute to the Responder phenotype are contained in the genome. Additionally, it is envisaged in accordance with the present invention that the nucleic acid molecules of the invention exert or enhance the Responder phenotype in conjunction with further sequences comprised, for example, in the T66A, T66B and T66C regions. Preferably, said additional regions encode MARK-related kinases.

Also, the person skilled in the art will, on the basis of the teachings of the present invention, be in a position to genetically manipulate the nucleic acid contributing to the Responder phenotype. He will further be in the position to screen the genome of an organism or cell of interest for additional nucleic acid sequences encoding Responder functions on the basis of the genetic organization of the Responder taught in accordance with the present invention. All these embodiments that are without further ado derivable from the specific teachings provided herein are also comprised by the present invention.

It is further envisaged in accordance with the present invention that the Responder may act as a component of a signaling cascade involved in sperm motility and/or the fertilization of oocytes. The t-Responder may act such as to protect the sperm carrying the t-form of the Responder from the negative actions of the t-Distorters whereas the sperm carrying the wild type form of the Responder is “poisoned” (see Lyon 1986). Therefore, the action of the t-form of the Responder somehow counteracts the t-Distorter function suggesting that the Distorters are part of the same signaling cascade. It is, thus, envisaged that the wild type gene or the products of any member of that signaling cascade, once molecularly known, can be manipulated such as to “poison” the sperm expressing either dominant active or dominant negative forms, or by overexpressing, reducing or abolishing the gene function of any member of said signaling cascade. Selection of genetic traits may then be easily achieved by manipulating the amino acid sequence, activity or expression level of any member of that signaling cascade and restricting the expression of the manipulated form preferentially or completely to those sperm carrying it, such as is the case for the Responder function. The promoter of the Responder or other promoters activating gene expression during the haploid phase of spermatogenesis would be a suitable means for achieving this restriction.

Accordingly, the present invention also relates to methods of influencing transmission ratio by manipulating the expression level or the protein activity of any other member of said signaling cascade. For the purposes of this invention, said cascade is termed “Responder/Distorter signal cascade”. It is further envisaged in accordance with the present invention that other signaling cascades may exist besides the Responder/Distorter signaling cascade that may be involved in the motility and/or fertilization capability of spermatozoa. Thus, it is envisaged in accordance with the present invention that the expression level and/or activity of one or more of the proteins involved in said other signaling cascades may be also manipulated in order to influence the transmission ratio. Influencing transmission ratio implies that said ratio may be enhanced or reduced. Methods for manipulating said expression level or said protein activity are known in the art and comprise methods of manipulating amino acid sequences and/or, e.g., promoter strengths or expressing an inhibitor of any member of said signaling cascade. Alternatively, it is envisaged that the expression level may be modulated on the transcription level, the level of pre-mRNA processing, mRNA transport and/or stability, and/or the translation level. Preferably, the modification and/or replacement of elements does not alter the tissue specificity or the specificity for the developmental stage of the expression unit. It is also envisaged in accordance with the present invention that the genetic background of the host organism, the site of integration, and/or the number of integrated copies of a transgene construct may influence the expression efficiency of said transgene construct. Expression or activity of one or more of said members may (significantly) be altered or enhanced, (significantly) be reduced or abolished. Said members also include the Distorters. These methods of the invention can, either alone or in conjunction with other methods described below, advantageously be used for the generation of transgenic animals. Said transgenic animals provide a suitable assay system to test whether the above mentioned methods for manipulating said expression level or said protein activity were successful. Such a system is described in Example 6. Furthermore, said transgenic animals may be employed in any of the breeding schemes addressed below.

In another preferred embodiment of the invention, said nucleic acid molecule is a DNA molecule.

The deduction of the amino acid sequence from the nucleic acid sequence of the invention allows the conclusion that the polypeptide is the expression product that contributes to the Responder phenotype. However, it is not excluded that the mRNA contributes to or triggers said Responder phenotype. Also, it is envisaged in accordance with the present invention that the expression level, stage of expression during spermatogenesis or the copy number of said gene results in or contributes to the Responder phenotype. Therefore, in a preferred embodiment of the nucleic acid molecule of the invention said expression product is an RNA or a (poly)peptide.

A further preferred embodiment of the invention is a nucleic acid molecule, wherein said Responder function is the mouse-t-complex Responder function.

Although it is easily possible to identify mutated or wild-type Responders in animals other than the mouse on the basis of the genetic structure of the Responder that is provided in accordance with the present invention, the mouse t-complex Responder may find applications, for example in breeding, also when introduced into other animals. Specific applications of the Responder function are addressed herein below.

The invention further relates to a regulatory region of the gene corresponding to the nucleic acid molecule of the invention being capable of controlling expression of said nucleic acid molecule.

The term “corresponding” as used in accordance with the present invention also means that the gene comprises the nucleic acid molecule of the invention or fragments thereof.

The term “regulatory region” in the present application refers to sequences which influence the specificity and/or level of expression, for example in the sense that they confer cell and/or tissue specificity. Such regions can be located upstream of the transcription initiation site, but can also be located downstream of it, e.g., in transcribed leader sequences or in an intron.

The term “a regulatory region of the gene corresponding to the nucleic acid molecule” refers to a region with the above mentioned capabilities that controls expression of the bipartite nucleic acid molecule referred to herein also as a “gene”.

Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers.

Preferably, said regulatory region is a naturally occurring regulatory region or a genetically engineered derivative thereof.

More preferably, said regulatory region comprises or is a promoter. Said promoter is preferably tissue specific and confers expression, for example, during spermiogenesis.

The term “promoter” refers to the nucleotide sequences necessary for transcription initiation, i.e. RNA polymerase binding, and also includes, for example, the TATA box.

In one embodiment, said promoter is or comprises a minimal promoter. According to the present invention, promoters from other species can be used that are functionally homologous to the regulatory sequences or the promoter of the murine gene, or promoters of genes that display an identical pattern of expression, in the sense of being expressed in sperm cells. As has been outlined above, it is possible for the person skilled in the art to isolate with the help of the known murine nucleic acid corresponding genes from other species, for example, human. This can be done by conventional techniques known in the art, for example, by using the nucleic acid molecule of the invention as a hybridization probe or by designing appropriate PCR primers. It is then possible to isolate the corresponding promoter region by conventional techniques and test it for its expression pattern. For this purpose, it is, for instance, possible to fuse the promoter to a reporter gene, such as the lacZ gene or green fluorescent protein (GFP) and assess the expression of the reporter gene in transgenic mice.

The present invention also relates to the use of promoter regions which are substantially identical to the murine promoter or to a promoter of a homologous gene or to parts thereof and which are able to confer specific expression in sperm cells.

Such promoters differ at one or more positions from the above-mentioned promoters but still have the same specificity, namely they comprise the same or similar sequence motifs responsible for the above described expression pattem. Preferably such promoters hybridize to one of the above-mentioned promoters, most preferably under stringent conditions. Particularly preferred are promoters which share at least 85%, more preferably 90-95%, and most preferably 96-99% sequence identity with one of the above-mentioned promoters and have the same specificity. Such promoters also comprise those which are altered, for example by deletion(s), insertion(s), substitution(s), addition(s), and/or recombination(s) and/or any other modification(s) known in the art either alone or in combination in comparison to the above-described nucleotide sequence. Methods for introducing such modifications in the nucleotide sequence of the promoter of the invention are well known to the person skilled in the art. It is also immediately evident to the person skilled in the art that further regulatory sequences may be added to the promoter of the invention. For example, transcriptional enhancers and/or sequences which allow for induced expression of the promoter of the invention may be employed. A suitable inducible system is for example tetracycline-regulated gene expression as described, e.g., by Gossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen et al. (Trends Biotech. 12 (1994), 58-62).

Most preferably, said regulatory region comprises the fragment from nucleotides 930 to 3576 of the sequence shown in FIG. 11.

Also comprised are fragments or variants of the above sequence wherein the regulatory function of said fragments or variants is essentially retained or even improved. This may be tested according to methods well known in the art in combination with the teaching of this specification.

The invention further relates to a recombinant DNA molecule comprising a nucleic acid molecule of the invention and/or a regulatory region of the invention and/or a regulatory region allowing expression during spermatogenesis/spermiogenesis.

Accordingly, the regulatory region may control expression of the nucleic acid molecule contributing to the Responder function. Alternatively, said recombinant DNA molecule may comprise said regulatory region which controls expression of a heterologous nucleic acid or which is not operatively linked to any nucleic acid and, thus, may be used for cloning purposes. In the first alternative, said regulatory region is operatively linked to a heterologous DNA sequence. For example, said regulatory region may be operatively linked to a naturally occurring or in vitro engineered DNA encoding a member of the Responder/Distorter cascade, for example, a Distorter or a member of another signaling cascade involved in sperm motility and/or fertilization. Also, in this embodiment of the invention, the nucleic acid molecule of the invention may be operatively linked to a different or to no regulatory region. The regulatory region may be the original regulatory region of the gene corresponding to the nucleic acid molecule of the invention or may be derived from a different copy of said gene or from a different gene. Furthermore, the regulatory region may be derived from a copy of the homologous gene (in case more than one copy exists) from a different species or may be derived from a different gene from said different species. The above-mentioned regulatory regions may also be modified in order to obtain optimum expression, which may be enhanced or reduced expression. Thus, it is envisaged in accordance with the present invention that e.g., the regulatory regions controlling expression of the gene comprising the T66k-20-cDNA (see FIGS. 7i, j, and k) or the cDNAs shown in FIG. 10 are used in unmodified or modified form in accordance with the present invention. Due to the teaching of the present invention, namely the cloning and the disclosure of the sequences of the cDNAs, it is routine experimentation for the person skilled in the art to clone and use said regulatory regions.

Advantageously, the recombinant DNA molecule of the invention may further comprise an expression unit encoding and expressing a desired genetic trait. Such a DNA molecule may be used to reduce, or enhance the inheritance of said desired genetic trait, provided that either the recombinant DNA molecule further comprises an expression unit encoding and expressing at least one Distorter or protein with Distorter activity, preferably D2, or the genetic background of the host provides such Distorter activity which may be naturally occurring in said host or which may have been introduced.

A particularly preferred embodiment of the invention relates to a recombinant DNA molecule, wherein said heterologous DNA sequence encodes a peptide, protein, antisense RNA, sense RNA and/or ribozyme. As regards the antisense RNA, it may find applications in methods of antisense therapy or antisense knockout strategies. Antisense therapy may be carried out by administering to an animal or a human patient, a recombinant DNA containing the regulatory sequences of the invention operably linked to a DNA sequence, i.e., an antisense template which is transcribed into an antisense RNA. The antisense RNA may be a short (generally at least 10, preferably at least 14 nucleotides, and optionally up to 100 or more nucleotides) nucleotide sequence formulated to be complementary to a portion of a specific mRNA sequence. Standard methods relating to antisense technology have been described (Melani, Cancer Res. 51 (1991), 2897-2901). Following transcription of the DNA sequence into antisense RNA, the antisense RNA binds to its target mRNA molecules within a cell, thereby inhibiting translation of the mRNA and down-regulating expression of the protein expected to be encoded by the mRNA. For example, an antisense sequence will be complementary to a portion of or all of the mRNA. In addition, ribozymes may advantageously be employed to eliminate wild-type Responder transcripts from cells.

The invention further relates to a recombinant DNA molecule, wherein said peptide, protein, antisense RNA, sense RNA, a toxin and/or ribozyme is capable of causing cell death.

In this embodiment of the invention, sperm which do not carry the R related transgene can be genetically selected.

For example, the promoter of the R gene can be used for the expression of a gene product inducing the destruction or apoptosis of said spermatocytes carrying said construct. Integration of such a construct on the X- or Y-chromosome will result in the transmission of the respectively other sex chromosome. Integration of the construct on the X chromosome will lead to the neutral transmission of the construct in female animals. Integration in the Y chromosome should, preferably, be in an inactive state that can be activated along the rules that will be laid down herein below.

A recombinant DNA molecule which further comprises DNA encoding an effector polypeptide is a further preferred embodiment of the invention.

It is particularly preferred that said effector polypeptide is capable of sequestering an ion selectively binding to a solid support, or binding to a preselected antigenic determinant or is a toxin, an enzyme, a ribozyme, a label or a remotely detectable moiety.

In accordance with the invention, it is most preferred that said effector polypeptide is calmodulin, methallothionein, a fragment thereof, green fluorescent protein (GFP), β-lactamase (Zlokamik et al., 1998), hCD24, myc, FLAG, hemagglutinin or an amino acid sequence rich in at least one of glutamic acid, aspartic acid, lysine, histidine or arginine.

Accordingly and in other words, the above embodiments of the invention relate to the use of the R promoter for the expression of a (poly)peptide being or having a tag. Said tag may be expressed in the cytoplasm of sperm. An example of such a tag is GFP or β-lactamase. Said tag is alternatively located on the surface of sperm and thus, may be recognized by specific antibodies. This enables the separation of sperm carrying a transgene expressed under the control of the R promoter from sperm not carrying said transgene. The person skilled in the art is familiar with a variety of methods for the separation of sperm carrying said tag on its surface. Preferably, said tag is selected by affinity chromatography or by using a cell sorter. After separation, sperm carrying the transgene or sperm without the transgene can be used for fertilization of eggs. This embodiment includes integration of transgene in either autosomes or sex chromosomes. Advantageously, the solid support referred to above is a membrane or the surface of an ELISA plate.

Further, the invention relates to a vector comprising the nucleic acid molecule of the invention, the regulatory region of the invention or the recombinant DNA molecule of the invention.

The vector of the invention may simply be used for propagation of the genetic elements comprised therein. Advantageously, it is an expression vector and/or a targeting vector. Expression vectors such as Pichia pastoris derived vectors or vectors derived from viruses such as CMV, SV-40, baculovirus or retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the recombinant DNA molecule or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors; see, for example, the techniques described in Sambrook, loc. cit. and Ausubel, loc. cit. Alternatively, the recombinant DNA molecules and vectors of the invention can be reconstituted into liposomes for delivery to target cells.

It is preferred according to one further embodiment that said vector comprises a heterologous promoter.

Said heterologous promoter not naturally operatively linked with the nucleic acid contributing to the Responder function may be used to determine a certain time point of the onset of Responder expression. This time point may be the same or a different one that is set when the natural Responder transcription unit is employed. For example, said heterologous promoter may also be active in the early or late haploid phase of spermatogenesis.

It is particularly preferred that said heterologous promoter is controlling gene expression in spermatogenesis and/or in spermiogenesis.

Most preferably, said heterologous promoter is the testis promoter of c-kit or of Angiotensin-Converting-Enzyme (ACE), both of which are well known in the art.

The invention further relates to a host cell transformed or transfected with the nucleic acid molecule, the recombinant DNA molecule or the vector of the invention. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Prokaryotic host cells will usually only be employed for the propagation of the nucleic acid molecule of the invention and sometimes for the production of the expression product. Suitable mammalian, fish or bird cell lines are well known or can easily be determined by the person skilled in the art and comprise COS cells, Hela cells, primary embryonic cell lines etc.

The term “transfected or transformed” is used herein in its broadest possible sense and also refers to techniques such as electroporation, infection or particle bombardment.

Furthermore, the invention relates to a method of recombinantly producing an expression product as defined herein above comprising the steps of culturing the host cell of the invention under conditions to cause expression of the protein and recovering said protein from the culture.

The method of the invention is most advantageously carried out along conventional protocols which have been described, for example, in Sambrook, loc. cit.

The invention further relates to an expression product encoded by the nucleic acid molecule of the invention or which is obtainable by the production method of the invention.

In accordance with the invention, said expression product may either be an mRNA or a polypeptide. Said expression product is, in accordance with the present invention, involved in the Responder phenotype and contributes to the phenomenon of transmission ratio distortion.

A further embodiment of the invention relates to an antibody specifically recognizing the expression product of the invention.

The antibody of the invention may be a monoclonal antibody or an antibody comprised in a polyclonal serum. Accordingly, the term “antibody” as used herein also relates to a polyclonal antiserum. In addition, said term relates to antibody fragments or fusion proteins comprising antibody binding sites such as Fab, Fv, scFv fragments etc. The antibody of the invention has a number of applicabilities including purification or diagnostic processes.

The invention additionally relates to a nucleic acid molecule specifically hybridizing with the nucleic acid molecule of the invention translatable into said MARK related kinase or to an intron of said nucleic acid molecule or with the regulatory region of the invention or with a complementing strand thereof.

Said nucleic acid molecules comprise at least 15 nucleotides in length and hybridize specifically with a nucleic acid or regulatory sequence as described above or with a complementary strand thereof. Specific hybridization occurs preferably under stringent conditions and implies no or very little cross-hybridization with nucleotide sequences having no or substantially different regulatory properties. Such nucleic acid molecules may be used as probes and/or for the control of gene expression. Nucleic acid probe technology is well known to those skilled in the art who will readily appreciate that such probes may vary in length. Preferred are nucleic acid probes of 17 to 35 nucleotides in length. Of course, it may also be appropriate to use nucleic acids of up to 100 and more nucleotides in length. The nucleic acid probes of the invention are useful for various applications. On the one hand, they may be used as PCR primers for amplification of regulatory sequences according to the invention. In this embodiment, one of the primers may hybridize to the 3′ portion of the Responder having a high homology to the rsk3 gene. Another application is the use as a hybridization probe to identify regulatory sequences hybridizing to the regulatory sequences of the invention by homology screening of genomic DNA libraries. Nucleic acid molecules according to this preferred embodiment of the invention which are complementary to a regulatory sequence as described above may also be used for repression of expression of a gene comprising such regulatory sequences, for example due to an aniisense or triple helix effect or for the construction of appropriate ribozymes (see, e.g., EP-B1 0 291 533, EP-A1 0 321 201, EP-A2 0 360 257) which specifically cleave the (pre)-mRNA of a gene comprising a regulatory sequence of the invention. Selection of appropriate target sites and corresponding ribozymes can be done as described for example in Steinecke, Ribozymes, Methods in Cell Biology 50, Galbraith et al. eds Academic Press, Inc. (1995), 449-460. Furthermore, the person skilled in the art is well aware that it is also possible to label such a nucleic acid probe with an appropriate marker for specific applications, such as for the detection of the presence of a nucleic acid molecule of the invention in a sample derived from an organism.

The above described nucleic acid molecules may either be DNA or RNA or a hybrid thereof. Furthermore, said nucleic acid molecule may contain, for example, thioester bonds and/or nucleotide analogues, commonly used in oligonucleotide anti-sense approaches. Said modifications may be useful for the stabilization of the nucleic acid molecule against endo- and/or exonucleases in the cell. Said nucleic acid molecules may be transcribed by an appropriate vector containing a chimeric gene which allows for the transcription of said nucleic acid molecule in the cell. Such nucleic acid molecules may further contain ribozyme sequences which specifically cleave the (pre)-mRNA comprising the regulatory sequence of the invention. Furthermore, oligonucleotides can be designed which are complementary to a regulatory sequence of the invention (triple helix; see Lee, Nucl. Acids Res. 6 (1979), 3073; Cooney, Science 241 (1988), 456 and Beal et al., Science 251 (1991), 1360), thereby preventing transcription and the production of the encoded mRNA and/or protein.

Furthermore, the invention relates to a pharmaceutical composition comprising the DNA molecule, the regulatory region, the recombinant DNA, the vector, the host cell, the expression product or the antibody of the invention.

Said pharmaceutical composition comprises at least one of the aforementioned compounds of the invention, either alone or in combination, and optionally a pharmaceutically acceptable carrier or excipient. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Compositions comprising such carriers can be formulated by conventional methods. These pharmaceutical compositions can be administered to subject in need thereof at a suitable dose. Administration of the suitable compositions may be effected by different ways, e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical, intradermal, intranasal or intrabronchial administration. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. A typical dose can be, for example, in the range of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression in this range); however, doses below or above this exemplary range are envisioned, especially considering the aforementioned factors. Generally, the regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg units per kilogram of body weight per minute, respectively. Progress can be monitored by periodic assessment. Dosages will vary but a preferred dosage for intravenous administration of DNA is from approximately 10⁶ to 10²² copies of the nucleic acid molecule. The compositions of the invention may be administered locally or systematically. Administration will generally be parenterally, e.g., intravenously; DNA may also be administered directly to the target site, e.g., by biolistic delivery to an internal or external target site or by catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Furthermore, the pharmaceutical composition of the invention may comprise further agents such as interleukins or interferons depending on the intended use of the pharmaceutical composition.

It is envisaged by the present invention that in particular the various recombinant nucleic acid/DNA molecules and vectors of the invention are administered either alone or in any combination using standard vectors and/or gene delivery systems, and optionally together with an appropriate compound and/or together with a pharmaceutically acceptable carrier or excipient. Subsequent to administration, said molecules may be stably integrated into the genome of the mammal, fish or bird. On the other hand, viral vectors may be used which are specific for certain cells or tissues, preferably for pancreatic cells and persist in said cells. Suitable pharmaceutical carriers and excipients are well known in the art.

The invention further relates to a diagnostic composition comprising the nucleic acid molecule, the regulatory region, the recombinant DNA molecule, the vector, the host cell, the expression product or a primer or an oligonucleotide hybridizing to the nucleic acid molecule or regulatory region of the invention or to a complementary strand thereof and preferably to the regions identified herein above or the antibody of the invention. Comprised by the above definition of the term “primer” are also pairs of primers such as forward and reverse primers that may be used for PCR. One of said primers of said pair of primers may hybridize in the region of the rsk-related nucleic acid sequence.

In one embodiment, said diagnostic composition is manufactured in the form of a kit.

Said compositions may additionally contain further compounds such as plasmids, antibiotics and the like for screening animals or cells for the presence of nucleic acid sequences or regulatory elements corresponding to those identified in the appended examples or described herein above. The components of the diagnostic composition and/or kit of the present invention may be packaged in containers such as vials, optionally in buffers and/or solutions. If appropriate, one or more of said components may be packaged in one and the same container. Additionally or alternatively, one or more of said components may be adsorbed to a solid support such as, e.g., a nitrocellulose filter or nylon membrane, or to the well of a microtiter plate.

The invention further relates to a method for the production of a transgenic non human mammal, fish or bird comprising introducing the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention into a cell, preferably germ cell, embryonic cell or an egg cell or a cell derived therefrom.

Methods for the generation of such transgenic animals are well known in the art and are described, for example, in “Guide to techniques in mouse development” (ed. Wassarman & DePamphilis) Methods in Enzymology Vol. 225 (Academic Press, 1993). The method of the invention also comprises embodiments related to the cloning of such animals. These embodiments include the steps of introducing said nucleic acid molecule, recombinant DNA molecule or vector of the invention into the nucleus of a cell, preferably an embryonic cell, replacing the nucleus of an oocyte, a zygote or an early embryo with said nucleus comprising said nucleic acid molecule, recombinant DNA molecule or vector of the invention, transferring either said ooyte, zygote or early embryo into a foster mother or first in vitro or in vivo culturing said oocyte, zygote or early embryo and subsequently transferring the resulting embryo into a foster mother and allowing the embryo to develop to term; see, for example, Wilmut I. et al. (1997) “Viable offspring derived from fetal and adult mammalian cells”, Nature 385, 810-813.

In a preferred embodiment of the method of the invention, said chromosome is an X chromosome or the corresponding sex chromosome in birds or fish or an autosome.

In an alternative preferred embodiment of the method of the invention, said chromosome is a Y chromosome, or the corresponding sex chromosome in birds or fish.

It is particularly preferred that the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention, a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined hereinabove alone or in combination is/are integrated in said Y chromosome in a reversible inactive state of expressibility.

In accordance with the method of the invention, it is most preferred that said nucleic acid molecule, regulatory region, recombinant DNA molecule, vector of the invention, a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined hereinabove alone or in combination is/are flanked by lox P sites or FRT sites.

In all the above embodiments, at least one Distorter may be present on the same or on different chromosome.

An additional particularly preferred embodiment of the method of the invention further comprises introducing a nucleic acid molecule encoding at least one Distorter into the same or a different chromosome or introducing a chromosomal fragment comprising at least one Distorter into said cell. Advantageously, said Distorters are the mouse t-complex Distorter loci.

It is most preferred that said Distorter is/are D2 and/or D1.

Said method of the invention and its various preferred embodiments provide a wide range of applications in particular in the breeding of animals. Thus, as has been outlined above, the nucleic acid sequence encoding a molecule contributing to the Responder and/or an effector (poly)peptide as defined hereinabove may be under the regulation of the promoter naturally associated with said nucleic acid sequence. Integration of such a construct into a chromosome will, in the absence of a Distorter function result in a disadvantage in a chromosome if it comes to transmission of said chromosome. This disadvantage may be in the range of 49 to 0% transmission ratio. In the case that the Responder effect results in a very low or no transmission of the corresponding chromosome and if, in addition, the above recited construct comprising the nucleic acid molecule of the invention or the effector (poly)peptide is integrated into the Y chromosomes, the Y chromosome and the Responder function would hardly or not be transmitted by male animals. In order to provide for male animals, the Y chromosome should advantageously comprise an inactive construct that can, however, be activated. Said inactive construct should be without influence on the transmission ratio. One embodiment of said construct comprises loxP or FRT sites which flank an intervening sequence located between said promoter or a heterologous promoter controlling expression in spermiogenesis and effector (poly)peptide encoding sequences and/or sequences conferring Responder activity. The intervening sequence would be designed in such a way as to prevent the expression of effector and/or Responder activity. Activation of the effector and/or Responder activity may be effected by excision of the intervening sequence due to activity of the Cre or flp protein comprised in the same cell. Another embodiment of said construct comprises loxP or FRT sites flanking said promoter or a heterologous promoter controlling expression in spermiogenesis whereby the promoter is oriented away from the construct comprising the nucleic acid of the invention or the effector sequences encoding the above mentioned (poly)peptides. The activity of Cre or flp would allow the promoter to be inverted resulting in the transcription of the effector sequences or the sequences contributing to Responder activity during spermiogenesis. Another embodiment of said construct comprises loxP or FRT sites flanking said nucleic acid sequences reversely oriented towards the promoter such that the antisense strand is transcribed during spermiogenesis. Activation may be effected by flipping the effector sequences or the sequences contributing to Responder activity due to the activity of Cre or flp comprised in the same cell. Expression of the Cre or flp protein would advantageously be effected prior to spermiogenesis. The activation of the Responder or effector function is in such cases effected during spermatogenesis under the control of the R promoter or another promoter controlling expression during spermatogenesis/spermiogenesis. Preferably, the Cre gene is integrated on an autosome and may be expressed under the control of one of the following promoters: cytomegalovirus immediate early enhancer-chicken beta-actin hybrid (CAG) promoter, wherein site specific recombination occurs in the zygote; adenovirus Ella promoter, wherein expression is triggered during early embryogenesis; CMV, wherein expression is triggered during embryogenesis; OCT4, wherein expression is also triggered during embryogenesis and in germ line cells; HSV-TK or Pgk, wherein expression is ubiquitous; or Pgk2, wherein the construct is expressed during spermatogenesis. In the above embodiment, the Responder and/or effector encoding construct is transmitted by male animals in an inactive state. Mating with a female carrier of the Cre construct will result in male progeny having their Responder and/or effector activated during spermatogenesis. Progeny of these male animals inherit predominantly or exclusively the X chromosome of the father and are accordingly female progeny. In the case that the X chromosome is exclusively transmitted, the Responder and/or effector function is not inherited by the progeny. However, in cases of a less strong effect of the Responder and/or effector (poly)peptide leading to, for example, 10 to 20% transmission, the inactivation of the construct is not necessary because this low transmission is sufficient for the generation of male carriers. The frequency of inheritance of the R gene of the mouse, without the interaction of t-Distorters, is naturally in the range of about 20%.

In an alternative preferred embodiment of the method of the invention that has been identified above, the Responder and/or effector is integrated on the X chromosome or on an autosome. In this case, no inactive construct is necessary, since the Responder and/or effector encoding construct is transmitted in female animals in a neutral state, because Responder function only acts during spermatogenesis. Mating with wild type male animals leads to the generation of male animals carrying an active R and/or effector encoding gene on the X chromosome or an autosome. The chromosome carrying the R and/or effector encoding gene has a disadvantage in transmission. This means less than 50% to 0% of the progeny inherit said chromosome. In the case that the R and/or effector encoding construct is integrated into the X chromosome, no female progeny or only a low percentage of female progeny will be generated.

Furthermore, the invention relates to a method for the production of a male transgenic non human mammal, fish or bird having integrated in its Y or corresponding sex chromosome the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention, a heterologous promoter controlling expression in spermiogenesis and/or a DNA sequence encoding an effector (poly)peptide as defined hereinabove alone or in combination in an active state of expressibility, said method comprising in vitro fertilization or injection of spermatozoa into eggs using sperm from said male transgenic non human mammal, fish or bird. In a preferred embodiment of the present invention, said method prior to in vitro fertilization or injection further comprises allowing expression of said effector (poly)peptide and selecting for sperm expressing said effector (poly)peptide and, thus, containing said Y or corresponding sex chromosome. The above method is useful in case the transmission of the construct from male carriers by natural mating or artificial insemination is close to 0%. The production of transgenic male carriers can be achieved by the method of the invention using in vitro fertilization since it has been shown in mice that transmission ratio distortion of t/+ sperm does not occur during in vitro fertilization. The efficiency of the method of the invention can be further enhanced by selection for sperm carrying a Y or corresponding sex chromosome prior to in vitro fertilization as described above. Selection can be effected, e.g., by cell sorting.

Alternatively, male carriers of the R and/or effector function which are used for the generation of predominantly female progeny result from mating of hemizygous male animals carrying an inactive R and/or effector encoding construct with hemizygous female animals carrying a locus encoding a site specific recombinase and preferably the Cre locus. Progeny of such matings may be used for the maintenance of the strain as well as for the generation of the desired female progeny. It is worthwhile noting that from a single male carrier of the R and/or effector encoding construct many female progeny can be obtained.

A further embodiment of the invention that has been referred to above relates to the use of the R gene in combination with Distorter 2 (D2) preferably in combination with Distorter 1 (D1). In this embodiment, the chromosome carrying the R construct is transmitted predominantly or exclusively.

Distorters D1 and D2 (and possibly D3 as well as further postulated Distorters) act in trans to the advantage of the chromosome carrying the R construct. Whereas the applicant does not wish to be bound by any scientific theory, it is presently assumed that D1 and D2 are expressed in the diploid phase of spermatogenesis. Whereas the Distorter genes have not yet been identified it is well known that their gene products lead to the predominant or exclusive transmission of the chromosome carrying the R function. The Distorter function can be provided, for example, by a chromosome carrying a partial t-haplotype containing, e.g., Distorter D1 or D2 or both. It is further presumed that the expression products of the Distorter genes exert a negative influence on sperm not carrying the R function. In contrast, the sperm carrying the R function are protected by the R function. It is also suggested that such sperm may have a selective advantage as regards motility and thus faster reach the egg cell to be fertilized.

It is envisaged in accordance with the present invention that D2, D1 and further Distorters are located on the same or one or more different chromosomes than that or those which carry/carries the R construct. If R is integrated on the Y chromosome, mating will predominantly result in male progeny. Integration on the X chromosome, in contrast, will yield predominantly or exclusively female progeny. Integration in an autosome will result in a high transmission of said chromosome and thus any trait linked to said R construct. The high transmission of the R construct guarantees the maintenance of the R function. A practical advantage of the embodiment, in the case that the R encoding construct is integrated in the X chromosome, is that only few male wild type animals are necessary for the maintenance of the Y chromosome, i.e., of the male sex. Said male wild type animals may be generated by mating transgenic hemizygous female animals, carrying both the Distorter(s) and the R function with wild type males.

The subject-matter of the invention relates also to a transgenic non human mammal, fish or bird having stably integrated in its genome the nucleic acid molecule, the regulatory region, the recombinant DNA molecule or the vector of the invention or which is regenerated from a host cell of the invention or which is obtainable by the method of the invention referred to above.

Said transgenic animal is advantageously mouse, cattle, sheep, pig, goat, rat, rabbit, horse, dog, cat, camel, chicken, duck, salmon or trout.

Said transgenic animals may be used for producing offspring at a non mendelian ratio comprising breeding, in vitro fertilization or artificial insemination.

The invention additionally relates to a pair of transgenic non human mammals, fish or bird, wherein the male is a transgenic animal having integrated in its Y chromosome the nucleic acid molecule, the regulatory region, the recombinant DNA molecule, or the vector of the invention in a reversible inactive state of expressibility and optionally at least one Distorter in its genome, and the female is a transgenic animal having stably integrated into its genomic DNA a nucleic acid molecule encoding a site specific DNA recombinase.

The pair of transgenic animals should of course be preferably of the same species in order to allow a successful mating.

Preferably, in said female of said pair of animals, said DNA recombinase is Cre or flp.

Most advantageously, said DNA recombinase is controlled by regulatory elements that are active prior to spermiogenesis.

Further, the present invention relates to sperm obtainable from a male of the transgenic non-human mammal, fish or bird as defined herein before. Said sperm may be comprised in a composition suitable, for example, for deep freezing.

The invention also relates to a method for the selection of the sperm of the invention comprising allowing expression of the effector (poly)peptide and selecting for the presence or absence of said (poly)peptide.

In accordance with this method of the invention, the effector (poly)peptide is preferably selected for by cell sorting or affinity chromatography. Sperm either carrying or not carrying the effector (poly)peptide and thus the nucleic acid molecule of the invention may then be used for the further desired purpose.

Additionally, the invention relates to a method for the selection against sperm of the invention comprising

(a) allowing expression of the recombinant DNA molecule defined herein above that is capable of causing cell death; and

(b) selecting for viable sperm.

Cell death can advantageously also be caused by the in vivo expression of an effector molecule comprising a tag and the addition of a specific antibody binding to the tag and of complement to sperm in vitro, resulting in the inactivation or lysis of the spermatozoa carrying the construct.

Said methods find applicability in cases where sperm carrying the R promoter function is to be selected against.

A further object of the invention is the use of the sperm for the production of offspring. Such a production may comprise breeding, in vitro fertilization or artificial insemination.

An additional object of the present invention relates to the use of the nucleic acid molecule of the invention, the regulatory region of the invention, the recombinant DNA of the invention, the vector of the invention, the host cell of the invention, the expression product of the invention or the antibody of the invention for the isolation of receptors on the surface of sperm recognizing attractants of the egg cell for the development and/or production of contraceptive.

Further, the present invention relates to the use of the nucleic acid molecule of the invention, the regulatory region of the invention, the recombinant DNA of the invention, the vector of the invention, the host cell of the invention, the expression product of the invention or the antibody of the invention for the identification of chemicals or biological compounds able to trigger the (premature) activation or inhibition (repression) of the signaling cascade in which the Responder function is envisaged to be involved in. Such compounds could be applicable as potent contraceptiva since it is envisaged that the activation or inhibition (repression) of said signaling cascade may affect the motility of sperm, due to rapid exhaustion of their energy reserve, and/or by inhibiting sperm movement and/or affect the ability of sperm to fertilize ovulated eggs.

The identification of said chemical or biological compounds could be achieved by standard screening technology using the activation of the wild type Responder protein expressed in cell culture cells as an assay. It is e.g. envisaged that activation of said protein may trigger microtubule disruption in cell culture cells similar to the effect obtained by overexpression of the MARK kinase. Compounds triggering or inhibiting such an effect could then be tested for their effect on the motility and/or fertilization ability of sperm. Alternatively, a similar screening system for said compounds could also be envisaged for sperm without prior employment of a screening assay in cell culture cells.

Furthermore, the nucleic acid molecule of the invention, the regulatory region of the invention, the recombinant DNA of the invention, the vector of the invention, the host cell of the invention, the expression product of the invention or the antibody of the invention can be used for the isolation of receptor molecules and/or other members of the Responder/Distorter signaling cascade to which said expression product which would be expected to be a (poly)peptide may bind. Said signal transducing molecules may be identified by immunoprecipitation of protein complexes involving the Responder (poly)peptide and cloning of the corresponding genes encoding them, or by Two Hybrid Screening techniques in yeast employing standard technology. In particular, most preferably the Responder gene or (poly)peptide may be used to isolate the membrane receptor of the signaling molecule which is envisaged to activate said Responder/Distorter signaling cascade. Said membrane receptor is envisaged to be most preferable as a target for the development of novel contraceptives.

Additionally, the present invention relates to a method for the detection of the nucleic acid molecule, the regulatory region, the recombinant DNA molecule, the vector, or the expression product of the invention or a different heterologous expression product encoded by said DNA molecule or vector in the transgenic non human mammal, fish or bird of the invention or a part thereof comprising identifying said nucleic acid molecule, regulatory region, recombinant DNA molecule or vector of the invention or a portion thereof in said transgenic animal or said part thereof. The method of the invention allows the identification of animals of the invention on the basis of the genetic constructs they carry in accordance with the invention. Moreover, the method allows the identification of such animals e.g. after slaughtering by analyzing parts thereof. It should be noted that sperm, egg cells and embryos are also to be considered as parts of said animals. Detection may be effected by PCR using primers specified herein above. Nucleic acid hybridization with a detectably labeled probe constitutes a different method of detection. It is further most important to note that any portion or component of the nucleic acid, recombinant DNA molecule or vector may be identified in accordance with the method of the invention as long as it is indicative thereof. Thus, for example, the vector may comprise a nucleic acid sequence without any biological function that is nevertheless indicative of said vector and thus, of the invention. In another embodiment the effector (poly)peptide may be used for detection. Of course, the nucleic acid molecule of the invention or a portion thereof may itself be detected. All embodiments conceivable by the person skilled in the art that comprise the above step underfall the method of the invention as long as they allow the detection of the above mentioned genetic material.

Also, the present invention relates to a method of distorting the transmission ratio of genetic traits comprising manipulating the sequence or expression level of a different member of the Responder/Distorter signal cascade than the t-Responder, and restricting the expression of the manipulated form of said different member preferentially or completely to those sperm carrying it.

Preferred embodiments and various applications of this method as well as methods of manipulating said sequence or expression level have been addressed herein before.

The invention also relates to a transgenic animal having a recombinantly manipulated altered sequence or expression level of a member of the Responder/Distorter signal cascade, and wherein the expression of said member has been restricted preferentially or completely to those sperm carrying it.

Preferably, said member of said signal cascade is not the Responder.

In these embodiments of the invention, the sequence or expression level of a preferably different member of the cascade than the Responder is altered or abolished. Simultaneously, it is expected that the activity of the Responder and/or one or more of the Distorters is affected. Depending on the type of alteration/abolishment of Responder/Distorter functions, these transgenic animals may be used in breeding schemes corresponding to the ones addressed above.

Finally, the present invention relates to a method for the distortion, to a non-Mendelian ratio, of the transmission of a genetic trait from male mammals to their offspring comprising expressing during spermatogenesis/spermiogenesis a gene involved in sperm motility and/or fertilization.

In a preferred embodiment of the invention said genetic trait determines the sex.

In another preferred embodiment of the method of the invention said gene is under the control of a promoter that allows expression during spermatogenesis/spermiogenesis.

The promoter may be the original promoter of said gene or may be derived from a different copy of said gene or from a different gene. Furthermore, the promoter may be derived from a copy of the homologous gene (in case more than one exists) from a different species or may be derived from a different gene from said different species. The promoters may also be modified in order to obtain optimum expression, which may be enhanced or reduced expression.

In a particularly preferred embodiment of the method of the invention said promoter allows the preferential or exclusive expression of said gene in sperm carrying said gene.

In a further preferred embodiment of the method of the invention said gene is engineered such as to interfere with the function of its wild type allele or with the function of other genes involved in sperm motility and/or fertilization, wherein said gene inhibits the function of one or more genes involved in sperm motility and/or fertilization, and/or wherein said gene causes cell death in spermatocytes/spermatids expressing it, and/or wherein said gene encodes a tag allowing the in vitro selection of sperm carrying said tag.

In a further preferred embodiment of the method of the invention said gene encodes an inhibitor of cAMP dependent protein kinase A.

In a particularly preferred embodiment said inhibitor is PKI or a functionally active derivative or fragment thereof.

As used in accordance with the present invention the term “functionally active derivative or fragment” denotes molecules that deviate from PKI by one or more amino acid substitutions, deletions, and/or additions but essentially retain the biologically activity/activities of PKI, i.e. retain at least the inhibitory activity on cAMP dependent protein kinase A. Examples of functionally active derivatives or fragments of PKI are well known to the person skilled in the art and can be found, e.g., in catalogues of biotechnology companies (see, e.g., the Promega catalogue of 1998).

In another embodiment, the present invention relates to a transgenic animal comprising a gene as defined hereinabove.

Finally, the present invention relates to a sperm obtainable from the transgenic animal of the present invention.

The references cited in the present specification are herewith incorporated by reference.

THE FIGURES SHOW

FIGS. 1a-1 b:

(a) The upper panel shows a schematical drawing of the extend of the t-chromosome region (thick bars) of complete and partial t-haplotypes on chromosome 17 of the mouse, as well as the mapping positions of the Responder (R^(t)) and two Distorters (D1, D2) contributing to the transmission ratio distortion phenomenon (TRD) in mice (Lyon 1984; Fox et al. 1985; Herrmann et al. 1986; Bullard et al. 1992). The Responder function maps to the T66B genomic region shown in more detail in the middle panel (Schimenti et al. 1987; Nadeau et al. 1989; Rosen et al. 1990; Bullard et al. 1992). The region carrying R is defined by the recombination breakpoints of the partial t-haplotypes t^(h44), t^(h51), t^(Jr1) which do not contain R^(t), and t^(h49) or t^(h2) which do contain R^(t). The breakpoints of t^(h2) and t^(h49) coincide (Bullard et al. 1992). The intervals within which the breakpoints must have occurred are not sharply defined (as indicated by broken lines); only t-haplotype DNA is indicated. The position of the marker Tu66 serves as an anchor point for correlating the mapping of the Responder with the genetic fine map shown on the lower panel. The genomic clones (cosmids cat.15, ct.184, ct.169, ct.195), restriction map and gene structure of the fusion of T66Bk and mouse rsk3 demonstrate that the Responder candidate T66Bk lies well within the region defined as carrying R^(t). The exon-intron structure of T66Bk has not been determined; black bars indicate restriction fragments containing exons of mouse rsk3 located in the T66B region (Kispert 1990). The fragments encoding T66Bk and T66Bk-2 sequences have been determined by hybridisation of α-³²P labelled fragment pCRt^(h2)-161/170 to cosmid DNA, restriction digested, electrophoresed and blotted onto Nylon membrane according to standard techniques and as described in figure legend 2, as well as by sequencing as described in figure legend 4.

(b) The analysis of the BamHI fragment B9.1 of cosmid cat.15 demonstrated that another T66Bk gene family member, T66Bk-2, is located on the centromere-close side of B9.1, whereas the telomere-close side contains the putative promoter and first exon of the T66Bk-rsk3 fusion gene. B9.1 contains the complete putative protein coding region on one exon and a single 3′-exon (indicated as 3′) encoding untranslated sequences of T66Bk-2. The putative promoter region and first exon encoding untranslated sequences of T66Bk-2 is located at the centromere-close side of B9.1 probably within the 6.1 kb BamHI fragment of cat.15, but the exact position has not been determined.

Methods:

The cosmids cat.15, ct.169, ct.184 and ct.195 were isolated from a cosmid library constructed from t^(w12)/t^(w12) genomic DNA prepared according to conventional techniques in the vector pcos2EMBL (Ehrich et al. 1987). Library screening and cosmid mapping were performed as described (Herrmann et al. 1987; Rackwitz et al. 1985; Kispert 1990). The restriction map as well as the structure and sequence of mouse rsk3 have been determined previously (mouse rsk3 was initially named Tck; Kispert, 1990). The chromosomal localization of genomic restriction fragments hybridizing to subfragments derived from cosmids or to cDNA probes was done by restriction fragment length polymorphism (RFLP) mapping (Fox et al. 1985; Herrmann et al. 1986). Polymorphic restriction fragments specific to t-haplotypes were assigned to the T66B region if present in genomic DNA from t^(h2), t^(h49), t^(low), t⁶, t^(w5) or other complete t-haplotypes, but not in DNA from t^(h44), t^(h51), or wild type inbred strains, according to previous characterizations of these t-haplotypes (Lyon 1984; Fox et al. 1985; Herrmann et al. 1986; Bullard et al. 1992).

FIG. 2:

Southern blot hybridization of genomic or cosmid DNA of various t-haplotype carrying mice, or wild type mouse strains. The DNA was digested with BamHI endonuclease, blotted on Nylon membrane and hybridized with the probe pCRt^(h2)-161/170. Two fragments, B7.8 and B9.1 (marked by an asterisk), are visualized in t-haplotypes carrying the Responder, but are absent from t-haplotypes without R function as well as from wild type strains. Both fragments are present in the cosmid cat.15 and together contain the transcription unit of the gene T66Bk, as shown on FIG. 1 (bottom left). B9.1 additionally contains the protein coding and 3′-untranslated region of T66Bk-2. A third hybridizing fragment on cosmid cat.15 of about 6.1 kb is likely to contain part of the T66Bk-2 gene. The 6.1 kb BamHI fragment is located at the proximal (centromere close) end of cosmid cat.15; it is truncated by the cloning event and thus, it is not identical in size with and cannot be correlated to any of the fragments identified in the hybridizations of total genomic DNA.

Abbreviations: t^(Jr1)=t^(w71Jr1); t^(low)=t^(lowH); T^(Or)=deletion chromosome T Oak Ridge 4. 129/Sv, C57BL/6 and DBA/2 are mouse inbred strains.

Methods:

Genomic DNA was prepared as described (Herrmann and Frischauf, 1987), digested with BamHI, blotted by an alkaline capillary transfer onto Hybond N+ membrane (Amersham) as described (Herrmann et al. 1986; Sambrook et al. 1989), UV treated in a UV Stratalinker 2400 (Stratagene) according to Church and Gilbert (1984), hybridized in 0.5M NaPi pH 6.8/7%SDS at 68° C. over night with 2×10⁶ cpm/ml of probe, washed in 40 mM NaPi pH 6.8/1%SDS at 68° C., and exposed on Kodak X-AR5 X-ray film and an intensifying screen at −80° C. The probe was prepared by random primer extension using the T7 QuickPrime kit (Pharmacia Biotech), 50 ng of probe DNA and 5 μl of α-³²P dCTP (Amersham) at 3000 Ci/mmole according to the suppliers instructions.

The cDNA probe fragment pCRt^(h2)-161/170 was prepared by standard PCR amplification in 20 mM Tris pH8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dATP/dCTP/dGTP/dTTP each, using 1 unit of the Taq DNA polymerase, approximately 50 ng of the cDNA pCRt^(h2)-161/144 as template, 20 pmole of primer 161 and 170 each. 15 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 2 minutes at 72°C. with a final extension of 5 min. at 72° C. were performed, the product was loaded on a 1% agarose gel in TAE buffer (Sambrook et al. 1989), electrophoresed, the amplified fragment cut out under long wave length UV light (366 nm) and purified by centrifugation through an EZ Enzyme Removers column (Amicon) and ethanol precipitation (Sambrook et al. 1989). The DNA was dissolved in TE.

FIGS. 3a-3 b:

RT-PCR analyses verify that T66Bk maps to the Responder region and is transcribed during spermiogenesis. a) RT-PCR of testis RNA with the primer pair 181/144 which is specific for the T66Bk-rsk3 fusion gene amplifies a cDNA fragment of 821 bp from RNA of t-haplotypes carrying the t-Responder (for comparison see FIG. 1) confirming that this gene is present in the t-Responder carrying region and is expressed in testis (upper panel). The quality of the RNA and first strand cDNA used for the assay was confirmed by RT-PCR with the primer pair 145/146 which amplifies a cDNA fragment of 769 bp from the mouse rsk3 gene (Tck, see Kispert 1990). The latter RT-PCR also produces a smaller fragment in t-haplotypes containing the T66B region, but not in wild type or t-haplotypes which do not contain the T66B region. This smaller cDNA fragment is due to the deletion of an exon in the T66B-copy of rsk3. A substantial level of transcription of the T66Bk-rsk3 fusion gene is first detectable in 22 days p.p. testis (lower panel). At this stage haploid spermatids have formed and are undergoing the transformation process into spermatozoa called spermiogenesis (Rugh 1990). The primer pair 155/170 amplifies a cDNA fragment of 815 bp derived from T66Bk as well as related genes. The presence of RNA at all stages of spermatogenesis tested with the primer pair 155/170 suggests an early onset of the transcription of one or several members of the T66Bk gene family. A very low (basal) level of transcript from the T66Bk-rsk3 fusion gene is also detectable in early stages of spermatogenesis. b) Comparative RT-PCR of testis RNA with primer pairs specific for testis specific transcripts of angiotensin converting enzyme (ACE, Howard et al. 1990), c-kit (Rossi et al. 1992) and mouse protamine 1 (mP1, Peschon et al. 1987) allows a correlation of the transcription of the T66Bk-rsk3 fusion gene with that of known genes. The promoters of all three genes have been analyzed in transgenic mice (Langford et al. 1991; Albanesi et al. 1996; Peschon et al. 1987). mP1 is supposed to be transcribed in round, ACE and c-kit in elongating spermatids. Since, in our RT-PCR analysis the T66Bk-rsk3 fusion gene appears to be transcribed slightly later than ACE and c-kit we conclude that expression of the T66Bk-rsk3 fusion gene most likely commences in elongating spermatids.

Methods:

Total RNA of testis tissue was prepared following homogenization of the tissue in LiCl/urea according to a published procedure (Auffray and Rougeon 1980). After ethanol precipitation the RNA was dissolved in 50 μl 10 mM Tris-HCV1 mM EDTA pH7.6 (TE) per approximately 100 mg starting material. 2 μl total RNA (appr. 6 μg RNA) were used for cDNA synthesis with an oligo(dT) primer according to the instructions of the SuperScript plasmid cDNA synthesis kit of Gibco/BRL. After first strand synthesis the reaction was diluted to 50 μl with TE. For PCR amplification 0.5 μl of the first strand cDNA stock solution was added to 20 μl of the reaction mix containing 20 pmole of each primer, 20 mM Tris pH8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dATP/dCTP/dGTP/dTTP each, and 1 unit Taq DNA polymerase. Reaction mixes were overlayed with mineral oil and 35 cycles of 30 seconds at 94° C., 30 seconds at 50° C. and 30 seconds at 72° C. were performed using a PTC-100 thermocycler (MJ Research, Inc.). The reaction products were electrophoresed in 1% or 2% agarose gels, as applicable, containing 0.4 μg/ml ethidium bromide in TAE buffer (Sambrook et al. 1989), and photographed on a UV light box. The 1 kb ladder of Gibco/BRL was used as marker, as shown on the left margin of each photograph.

FIGS. 4a-4 c:

a and b) Nucleic acid (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:2) of pCRt^(h2)-161/144, representing a partial cDNA of the T66Bk-rsk3 fusion gene encoding a putative protein of 484 amino acid residues. Several in frame stop codons 5′ to the first methionine (start codon) and the stop codon at the end of the single long open reading frame suggest that the protein coding region of this cDNA is complete. However, the 5′ and 3′ non-coding sequences are most likely incomplete. An asterisk indicates the junction between the T66Bk gene and the truncated mouse rsk3 gene. Nucleic acid sequences of primers used for RT-PCR detection and cloning 30 of T66Bk sequences are indicated. The primer number and 3′ end are given.

c) Partial nucleic acid sequence (SEQ ID NO:3) of a cDNA fragment, ptlib0.7, consisting of a fragment from the 5′ end of a T66Bk-related gene fused to part of a mouse rsk3-related gene. This partial cDNA was isolated by PCR amplification with a plasmid vector anchor primer (seq5lib) and primer 144, from clone pools of a total of approximately 200,000 clones of a cDNA plasmid library constructed with RNA extracted from testis of a t^(w5)/t^(w12) adult male. Another 380,000 cDNA clones were screened by cDNA filter hybridization. From those clones another partial cDNA containing a sequence homologous to the one shown here, fused to rsk3 sequences, was obtained. A primer (161) located at the 5′ end of the cDNA sequence shown was designed and used in combination with primer 1.44 (rsk3) to amplify the cDNA fragment of T66Bk shown on FIGS. 4a and b, from testis cDNA of a t^(h2)/t^(h2) adult mouse.

Methods:

A cDNA library of testis RNA of an adult male carrying the complete t-haplotypes t^(w5)/tw¹² was constructed in the plasmid vector pSV-Sport1 using the SuperScript Plasmid cDNA synthesis kit (Gibco/BRL) according to the suppliers instructions. RNA isolation was performed as described in the legend to FIG. 3, mRNA purification was done using Oligotex beads according to the supplier's instructions (Qiagen). DNA preparations of library pools of a total of appr. 200,000 clones were prepared with the Qiagen plasmid midi kit (Qiagen) and tested by PCR amplification as described in figure legend 3 using primer pair seq5lib/144. A fragment of 0.7 kb was obtained and cloned in the vector pCR2.1 using the TA cloning kit of Invitrogen according to the instruction manual. Another 380,000 cDNA clones were plated on filters and screened by hybridization as described (Herrmann et al. 1987).

The partial cDNA pCRt^(h2)-161/144 was obtained by PCR amplification of cDNA, prepared and amplified as described in figure legend 3, except that the primer extension time at 72° C. was 2 minutes per cycle, from testis RNA of an adult male homozygous for the t-haplotype t^(h2), with the primer pair 161/144. The cDNA fragment was purified from a 1% agarose gel as described in figure legend 3, and cloned in the plasmid vector pCR2.1.

Plasmid DNA was prepared with the Qiagen Plasmid Midi kit. Sequencing reactions were performed using the RR DyeDeoxy Terminator Cycle Sequencing kit (PE Applied Biosystems) according to the instructions and gene specific primers (MWG Biotech) designed with the OLIGO Primer Analysis Software (NBI), the reactions were purified by centrifugation through Centri-Sep columns (Princeton Separations) according to the instructions, and run on an automatic ABI Prism 310 Genetic Analyzer (PE Applied Biosystems). Sequences were evaluated with the MacMolly Tetra programs set (Soft Gene, Berlin) on a Power Macintosh computer.

FIG. 5:

Northern blot hybridization demonstrating the transcription of T66Bk-gene family members. Transcripts are detectable in adult testis from all t-haplotype or wild type strains tested, but not in RNA from any other organ tested. During spermatogenesis a detectable level of transcript first appears at 22 days p.p. For a control the blot was re-hybridized with a probe for GAPDH (Kispert 1990).

Methods:

RNA was extracted as described (Auffray and Rougeon 1979), 10 μg per lane was loaded on a 1% agarose gel containing formaldehyde and electrophoresed in MOPS buffer according to standard techniques (Sambrook et al. 1989). The gel was washed twice for 20 minutes in 0.1M NH4-acetate, once in 50 mM NaPi buffer pH 6.8, in 2 gel volumes each, and blotted onto Hybond N+ membrane (Amersham) by capillary transfer (Sambrook et al. 1989) using a reservoir of 50 mM NaPi buffer pH 6.8. The filter was UV treated in a UV Stratalinker 2400 (Stratagene) according to Church and Gilbert (1984), hybridized with 2×10⁶ cpm/ml of the probe pCRt^(h2)-161/170 in 0.5M NaPi buffer pH 6.8/7%SDS/25% formamide at 68° C. over night, washed in 50 mM NaPi buffer pH 6.8/1%SDS at 68° C. and exposed on Kodak X-AR5 film using an intensifying screen. The probe fragment was amplified by PCR with the primer pair 161/170 using the cDNA pCRt^(h2)-161/144 as template and labeled as described in figure legend 2. To determine the relative amount of RNA in each lane the filter was re-hybridized as above with the cDNA clone pme66 containing a partial cDNA of the GAPDH gene (Kispert 1990).

FIG. 6:

Southern blot hybridization of DNA derived from several mammalian species and the chick, with the probe pCRt^(h2)-161/144 demonstrates the presence of T66Bk-related genes in hamster, rabbit, pig, human and chick suggesting the conservation of this gene class during evolution. The DNA was digested with BamHI, blotted on Nylon filter, and hybridized and washed at reduced stringency (58° C.).

Methods:

Genomic DNA was isolated from organs or blood cells (human) as described (Herrmann and Frischauf 1987), cut with BamHI endonuclease, electrophoresed in a 1% agarose gel in TBE buffer and blotted by alkaline capillary transfer as described (Sambrook et al. 1989; Herrmann et al. 1986) onto a Hybond N+ membrane (Amersham). The filter was UV treated in a UV Stratalinker 2400 (Stratagene) according to Church and Gilbert (1984), hybridized with 2×10⁶ cpm/ml of the probe pCRt^(h2)-191/144 in 0.5M NaPi buffer pH 6.8/7%SDS at 58° C. over night, washed in 100 mM NaPi buffer pH 6.8/1%SDS at 58° C. and exposed on Kodak X-AR5 film using an intensifying screen. The probe fragment pCRt^(h2)-161/144 was labeled as described in figure legend 2.

FIGS. 7a-7 c

The mouse genome contains several members of the T66Bk gene family. a and b) The protein coding exon of one member, T66Bk-2, is located in a tandem duplication arrangement on the centromere-close side of T66Bk, contained in the BamHI fragment B9.1 of the T66B region cosmid cat.15. The nucleotide (SEQ ID NO:4) and putative amino acid sequence (SEQ ID NO:5) of this exon are shown (FIGS. 7a and b). The sequence of primer 232 and 237 used for cDNA detection, mapping and expression studies (see FIG. 8) are indicated by a dashed line. A single base which is deleted in the cDNA T66k-8 (T 1164) is underlined.

c, d, and e) The cDNA T66k-8 was isolated from a testis cDNA library of the genotype t^(w5)/^(w12) The nucleotide (SEQ ID NO:6) and putative amino acid sequence (SEQ ID NO:7) are shown (FIGS. 7c, d, and e). Its nucleotide sequence is identical to that of T66Bk-2 in the region of overlap except for a single base deletion resulting in a shift of the open reading frame from amino acid residue 359 onwards (underlined). The sequences for primer 161 and 237 are indicated (see FIG. 8).

f, g, and h) SEQ ID NO:8: includes the cDNA T66k-7as is derived from an antisense transcript of a T66Bk family member. The 5′ end of T66k-7as is closely related to sequences upstream of the T66Bk promoter. Its 3′ end is very similar to the 5′ intron near the protein coding exon of T66Bk/T66Bk-2 (see FIGS. 7a and b). The location of T66k-7as in the genome has not been determined. Vector sequences are underlined by a dashed line, sequences with a high similarity to the exon encoding the large ORF of T66Bk/T66Bk-2 by a double dashed line, sequences with high similarity to intron sequences upstream or downstream of the protein coding and 3′-untranslated exon, respectively, of T66Bk/T66Bk-2 by “″”. The direction of transcription of the T66Bk/T66Bk-2 homology region is indicated.

i, j, and k) The cDNA clone, T66k-20, was isolated from the t^(w5)/t^(w12) testis cDNA library. The nucleotide (SEQ ID NO:9) and putative amino acid sequence (SEQ ID NO:10) shows a strong similarity to the above members of the T66Bk gene family.

l) Comparison of the putative amino acid sequences of the members of the 30 T66Bk gene family. Amino acid residues identical to T66Bk are indicated by “. Gaps indicated by_were introduced to allow optimal alignment. Note the strong similarity of all protein sequences as well as the altered protein tail in T66k-8. Note also the closer relationship of T66Bk-2 and T66k-20 compared to T66Bk, despite the fact that T66k-20 is longer at the N-terminus. T66Bk, SEQ ID NO:2; T66Bk-2, SEQ ID NO:5; T66Bk-20, SEQ ID NO:10; T66Bk-8, SEQ ID NO:7.

Methods:

The BamHI fragment B9.1 of cosmid cat.15 was isolated by restriction digestion and cloned in the vector pBluescript KS according to standard techniques. The DNA preparation and sequencing was carried out as described in Figure legend 4. The cDNA clones T66k-7as, T66k-8 and T66k-20 were isolated from a cDNA library constructed from testis of a t^(w5)/t^(w12) male, the library plated and screened by hybridization with a cDNA fragment derived from PCR amplification of the cDNA pCRt^(h2)-161/144 with the primer pair 155/170. Library screening, probe preparation, hybridization, plasmid preparation, sequencing etc. are described in figure legends 2, 3 and 4.

FIG. 8:

The T66Bk-2 gene is located in the T66B region and is expressed from 22 day p.p. in the testis.

A cDNA fragment of 951 bp derived by RT-PCR amplification of testis RNA and hybridization with a T66Bk-2/T66k-8 specific primer (232) is detectable in RNA derived from mice carrying the t-haplotypes t^(h2), t49, t⁶ and t^(w5) but not in t^(h44), t^(h51) and t^(Jr1). Therefore it maps to the T66B region, in agreement with the mapping data of cosmid cat.15. The signal obtained from t^(h2)/t^(h2) and t^(h49)/t^(h49) is higher than that obtained from T^(Or)/t⁶ or T^(Or)/t^(w5) in agreement with the fact the former two are homozygous for T66Bk-2, while the latter are heterozygous. A faint signal is obtained in t-haplotypes carrying the T66A region only or in wild type (Balb/c). This is due to a reduced capability of binding of the oligonucleotide 232 to other members of the T66Bk gene family. In testis RNA derived from t⁶/+ males of different stages (lower panel) T66Bk-2 transcription is first detected at 22 days p.p. However, the signal is very weak, but is significantly increased at 24 days p.p. This suggests that T66Bk-2 may be expressed at a lower level or later than T66Bk. Overall, the transcription level of T66Bk-2 in each testis sample detected by RT-PCR and hybridization correlates well with the number of T66Bk-2 alleles present in each of the samples. This together with the sequence conservation further suggests that the cDNA clone T66k-8 is derived from the locus T66Bk-2 within the T66B region.

Methods:

RNA derived from testis was reverse transcribed, first strand cDNA was amplified by PCR using the primer pair 161/237 (see FIGS. 7a and b, 7 c, d, and e), and the products separated by electrophoresis on 1% agarose as described in figure legend 3. The cDNA was transferred to Hybond N+ filters as described in figure legend 2, and hybridized with oligonucleotide 232 labeled using the DIG Oligonucleotide Tailing Kit (Boehringer Mannheim) according to the instructions of the supplier. Hybridization was carried out in 0.5M NaPi pH 6.8/7% SDS at 37° C. The filters were washed 4 times for 5 minutes in prewarmed 40 mM NaPi pH 6.8/1% SDS (37° C.) at room temperature. Prehybridization and oligonucleotide detection were done according to the protocol from Boehringer (Mannheim).

FIGS. 9a-9 b:

a and b) Nucleic acid (SEQ ID NO:11) and amino acid sequence (SEQ ID NO:12) of a cDNA encoding the T66Bk gene. The sequence extends the sequence of pCRt^(h2)-161/144 shown on FIGS. 4a and b, both at the 5′- and at the 3′-side, but is identical in the region of overlap. The 3′-end of the cDNA pSV-T66Bk ends in an intron of the mouse rsk3 gene and lacks a consensus polyadenylation signal suggesting that it was derived by oligo(dT) priming of incompletely spliced RNA. Asterisks indicate the positions of introns. The asterisk between position 2023 and 2024 indicates the fusion point between MARK- and rsk3-homology regions of T66Bk.

Methods:

Another cDNA library, in addition to the one used to isolate cDNAs presented on FIG. 7, was constructed from testis RNA of a male carrying the complete t-haplotypes t⁶/t^(w5) according to the methods described in figure legend 4 and screened as described in figure legends 7, 2, 3 and 4. A total of 500 000 cDNA clones contained in 10 pools were analysed by PCR for the presence of cDNA clones encoding the gene T66Bk using the primer pair 161/144. Four positive clones were identified and one, named pSV-T66Bk, was purified by colony hybridization screening using the cDNA pCRt^(h2)-161/144 as probe, and sequenced.

FIGS. 10a-10 f:

Nucleic acid and putative amino acid sequences of wild type members of the T66Bk kinase gene family.

a and b) The cDNA pCR.Balb-66k was isolated by RT-PCR from testis RNA of the wild type inbred mouse strain Balb/c. The putative start codon of the open reading frame is located 20 amino acid residues further upstream from the translation start of the T66Bk gene, very similar to the situation observed in T66k-20. The ORF is equal in length to that of T66k-20. Since in both genes, pCR.Balb-66k and T66k-20, the putative translation start does not conform closely with Kozak's rules it is possible that this start codon of translation is not efficiently used. Thus, it might be that either this or the next 3′-located translation start codon or both are utilized. The nucleotide (SEQ ID NO:13) and putative amino acid sequence (SEQ ID NO:14) are shown.

c and d) The cDNA pCR.C3H-66k was isolated by RT-PCR from testis RNA of the wild type inbred mouse strain C3H/N using the primers 161/220. In contrast to the ORF of T66Bk, the ORF of this gene is shorter at the C-terminal end resulting in a putative protein of 433 amino acid residues. The nucleotide (SEQ ID NO:15) and putative amino acid sequence (SEQ ID NO:16) are shown.

e and f) This is also the case for the ORF encoded by the genomic clone fragment pI.129-66k derived from the 129Sv wild type inbred mouse genome. The significance of this alteration of the ORF compared to the gene T66Bk is unclear. However, it is assumed that the length of the ORF and thus the resulting protein sequence may influence the properties of the protein. The nucleotide (SEQ ID NO:17) and putative amino acid sequence (SEQ ID NO:18) are shown.

FIGS. 11a-11 c:

Nucleic acid sequence (SEQ ID NO:19) of the putative promoter of the gene T66Bk. The BamHI fragment B9.1 of the cosmid cat.15 contains the protein coding region of T66Bk-2 (see FIGS. 2 and 7) as well as the putative transcription start site and upstream region of T66Bk. The sequence of 3641 bp presented here shows the intron and 3′-untranslated exon of T66Bk-2, located 3′ of the T66Bk-2 sequence shown on FIG. 7, followed by the upstream region and putative first exon of T66Bk. Splice donor/acceptor sites are indicated by an asterisk (*). Exon sequences are underlined. The underlined exon sequence of T66Bk shown represents the sequence contained in the cDNA pSV-T66Bk; the transcription start site of T66Bk, however, may be located further upstream. Two consensus TATA boxes are shown in bold type and underlined. The transcription start site of T66Bk has not been determined, but is likely to be located 3′ of either of the TATA boxes. It cannot be excluded that both TATA boxes are utilized alternatively for binding of the TATA binding protein complex. The restriction sites for KpnI and PmII used to isolate the putative promoter fragment utilized in the construction of tg5 are indicated in bold type. The sequence contains a number of potential binding sites for known transcription factors (Faisst and Meyer 1992). However, since none of them have been demonstrated to be functional, they have been omitted on the figure. Their positions can be readily identified by sequence analysis software such as MacMolly's Interpret program (Softgene, Berlin). Regulatory elements conferring tissue and stage specific regulation of transcription are often located just upstream of the transcription initiation sites, but may also be located in the first exon, intron or at a distance either far upstream or downstream. It is not known whether the sequence shown here contains all cis-regulatory elements or only a subset required for specific expression of T66Bk during spermiogenesis. It is also envisaged that the long 5′-untranslated region of T66Bk mostly comprised by exon 1 may have a function in regulating the onset and/or efficiency of translation.

Methods:

Cloning and sequencing of BamHI fragment B9.1 were done as described in figure legend 7.

FIGS. 12a-12 b:

The transgenes tg4 and tg5 are expressed during spermiogenesis.

To confirm that the transgenes tg4 and tg5 which showed distortion of their transmission from male carriers to their offspring are expressed in the testis, RT-PCR analysis was carried out using a transgene specific primer pair. For tg4 the primer pair 309/310 amplifying a junction fragment between the MARK- and rsk3 homology regions was used. For tg5, the primer pair 313/314 amplifies its 3′-end from hCD24 to the polyadenylation signal sequence. Various post partum stages of testis expected to be in the process of spermatid maturation were analyzed. mRNA was DNAsel treated before reverse transcription and 1 μl of this solution was amplified by PCR (+DNAsel/−RT). After reverse transcription of the remainder, 1 μl of it was amplified in parallel. Tg5-43 was tested with 313/314 except for tg5-43 stage 39 days p.p. which was control tested with the primer pair 309/310.

None of the control reactions showed a PCR product, whereas all samples subjected to reverse transcription yielded the expected fragment after PCR. This demonstrates the expression of tg4 and tg5, respectively, in the testes of male carriers. However, expression occurs earlier than expected from the analysis of c-kit and T66Bk shown on FIG. 3. This might be due to the sensitivity of the RT-PCR assay which might detect basal transcription of the transgenes, or to inappropriate control of transgene expression caused by the promoter fragment used in the construction or caused by influences of the integration sites. On the other hand, the adult male carrying tg4-3 and the tg5-43 39 day p.p. male showed a stronger fragment suggesting an increase of transgene expression during maturation or following mating to females. Abbreviations: ad, adult male (mated); M, marker (1 kb ladder (Gibco/BRL)

Methods:

RT-PCR was carried out essentially as described in figure legend 3 with the following exceptions. Before addition of Reverse Transcriptase to the reaction 1 μl of DNAsel (RNAse free, 10 units/μl) was added and the reaction was incubated at 37° C. for 20 min. 1 μl of the reaction was removed and kept on ice, to the remainder 1 μl of SuperscriptII Reverse Transcriptase (200 units/μl, Gibco/BRL) was added and the reaction was incubated for a further 20 min. each at 37° C. and 55° C. All PCR reactions were set up with the same PCR stock solution to which 1 μl of either the control reaction (+DNAsel/−RT) or the test reaction (+DNAsel/+RT) were added. PCR using the primer pair 309/310 was carried out as described in table 1 legend. The same conditions were used for the primer pair 313: 5′-ATGGGCAGAGCAATGGT-3′ (SEQ ID NO:22) and 314: 5′-CAGGTTCAGGGGGAGGT-3′ (SEQ ID NO:23).

FIG. 13:

T66Bk contains a second ORF encoding an N-terminal polypeptide of mouse rsk3.

The figure shows the cDNA sequence (SEQ ID NO:20) of pSV-T66Bk emphasizing the ORF encoded by the rsk3 homology region. The putative translation start and stop codons of the MARK-homology region as well as two potential translation start codons of the rsk3 homology region are underlined. The amino acid sequence (SEQ ID NO:21) shown starts at an ATG codon located 3′ of the stop codon of the MARK related kinase and 5′ of the splice site, indicated by an *. Another potential translation start codon is located in the rsk3 homology region. Although unlikely, there are two possibilities that this ORF is translated. First, the ribosome might not fall off the mRNA after completing translation of the MARK-related kinase and re-start translation at the next ORF. Second, altemative splicing might skip the exon encoding the MARK-related kinase. This would result in a transcript in which the ATG at position 2107-2109 would be the first potential translation start site. The latter is the case observed in the partial cDNA sequence ptlib0.7 shown on FIG. 4c demonstrating that such transcripts exist. However, they are not observed in males carrying the t-haplotypes t^(h2) or t^(h49), but only in complete t-haplotypes suggesting that they are derived from a gene located outside of the region carrying the t-Responder.

The examples illustrate the invention.

EXAMPLE 1 Cloning of a Novel Candidate Gene For The t Complex Responder

Cosmid clones from the T66B region were isolated and their genomic location within T66B verified by RFLP mapping (FIG. 1). In particular, the fragment pAK34 which is contained within the overlap of the cosmids ct.184 and cat.15 hybridizes to 3 genomic BamHI fragments in complete t-haplotypes, of which one, a 5.5 kb fragment, is located in the T66B region (Kispert 1990). The cosmids ct.184 and cat.15 contain the 5.5 kb BamHI fragment hybridizing to probe pAK34, thus confirming that they are derived from the T66B region. Likewise, the PCR fragment 161/170 derived from the cDNA described here hybridizes to the BamHI fragments B9.1 and B7.8 contained within cosmid cat.15, and both can be mapped to the T66B region (FIGS. 1 and 2).

A gene spanning at least 60 kb of the genomic region contained within the cosmid cluster isolated from the T66B region was identified. This gene is represented in 3 copies in t-haplotypes, one each in the regions T66A, T66B and T66C. The wild type form of it encodes the mouse homologue of human rsk3 (Zhao et al. 1995), a kinase of the pp90 ribosome S6 kinase family (called Tck in Kispert 1990). The gene copy located in the T66B region is altered compared to wild type (Kispert 1990). The 5′ end is not contained within cosmid cat.15 and one additional exon. is missing. The fact that one additional exon is missing was detected by RT-PCR of testis RNA derived from a panel of partial and complete t-haplotypes and wild type with the primer pair 145/146. In addition to the expected fragment of 769 bp a smaller fragment was obtained in the t-haplotypes containing the T66B region, but not in those containing only T66A nor in wild type. (FIG. 3a). This demonstrated that the T66B gene copy of rsk3 is expressed in testis. To identify the 5′ sequence of this gene, a cDNA library was constructed from mRNA of the testis of a t^(w5)/t^(w12) male mouse. Surprisingly, two clones were isolated from a total of approximately 580000 cDNA clones screened which contain heterologous sequences 5′ to base 438 of wild type rsk3 (Kispert 1990). The partial sequence of one of these clones is shown on FIGS. 4c. Primers for polymerase chain reaction (PCR) amplification were designed such that the forward primer (161) is located at the 5′ end of this cDNA, that is within the novel sequence, and the reverse primer (144) is located in the rsk3 sequence. PCR amplification of testis cDNA prepared from RNA of the partial t-haplotypes t^(h2) and t^(h49) produced a fragment of 2.1 kb, whereas no band was detected in t^(h44), t^(h51), t^(Jr1) or BALB/c (wild type) cDNA. The fragment (pCRt^(h2)-161/144) was isolated from t^(h2), cloned and sequenced (FIGS. 4a and b). It comprises yet another novel gene located within the T66B region (see below).

A primer pair (181/144) designed on the basis of the sequence of pCRt^(h2)-161/144 allows the amplification of a cDNA fragment of a testis expressed gene which is contained in t^(h2), t^(h49), t^(w5) and t⁶, but not t^(h44), t^(h51), t^(Jr1) or BALB/c (wild type) testis (FIG. 3a). Thus the corresponding transcript is t-specific and derived from a gene mapping to the T66B region. RT-PCR with the primer pair 145/146 for mouse rsk3 also confirmed the quality of the first strand cDNA synthesis. The cDNA-mapping by PCR confirms the genomic localization by Southern blot hybridization (see FIGS. 1 and 2).

EXAMPLE 2 The t Complex Responder Candidate Gene Encodes a Novel Kinase

The sequence of the 2.1 kb cDNA fragment pCRt^(h2)-161/144 contains a single long open reading frame (ORF) encoding a protein of 484 amino acid residues (FIGS. 4a and b). Several “in frame” stop codons upstream of the first potential translation start codon (bases 337-339) suggest that the N-terminal end of the putative protein is complete. The translation stop (bases 1789-1791) is still located within the “non-rsk3” sequence; the rsk3 sequence of the fusion transcript starts at base 1837.

Sequence comparisons with protein sequence databases revealed several known motifs within the ORF, most importantly a protein kinase domain and a consensus protein tyrosine kinase active site. However, the pattern of conserved residues is more strongly related to the consensus for serine/threonine kinases, suggesting that the isolated gene encodes a novel Serine/threonine kinase. However, the in vivo specificity remains to be determined experimentally. In accordance with the present invention, the gene is called T66Bk. The best match to known kinases was found to MARK, a recently published serine/threonine kinase which is involved in the regulation of the cytoskeleton (Drewes et al. 1997). The identity to MARK2 is more than 25% and approximately 38% at the amino acid level within the putative kinase domain. The putative protein contains 8 potential phosphorylation sites for casein kinase II, 5 for protein kinase C and 5 potential myristoylation sites.

The data explained above suggest that the T66Bk-rsk3 fusion gene arose by a rearrangement event resulting in the fusion of two gene parts, both derived from a kinase. The 5′ region probably including the transcriptional control elements are derived from a MARK related kinase. The 3′ end which is derived from the mouse rsk3 gene and may include most of its sequence and probably also its poly(A) addition signal might be around 5 kb long. The Southern blot hybridization data shown in FIG. 2 suggest that the genome may contain several gene family members of the MARK-related kinase.

EXAMPLE 3 Transcripts Derived from T66Bk-gene Family Members Accumulate During Spermiogenesis

In a Northern blot hybridization assay transcripts derived from T66Bk related genes can be detected in 22 day post partum (p.p.) male t⁶/+ testis or later, using the cDNA fragment pCRt^(h2)-161/170 as a probe (FIG. 5). Two transcripts of approximately 2.8 kb and 3.2 kb can be distinguished in T^(Or)/t⁶ and T^(Or)/t^(w5) testis RNA. Only the lower band is clearly detectable in BALB/c (wild type) testis RNA. This difference may be caused by differential splicing or different sequence of gene variants which distinguish, for example, t-haplotypes and wild type or various wild type strains. As the expected transcript size of the T66Bk-rsk3 fusion gene is appr. 7 kb, an assignment of one of the observed RNA bands to the T66Bk-rsk3 fusion gene is not possible. The Northern analysis showed that the members of the T66Bk gene family are fairly specifically expressed, and might even be restricted to the testis, as no transcripts were detected in RNA isolated from ovary, liver, spleen, kidney, lung or heart.

In a RT-PCR analysis of testis RNA using the primer pair 155/170, transcripts are detectable as early as day 7 p.p., the earliest stage of spermatogenesis tested (FIG. 3a). This suggests that low level transcription of one or several T66Bk-related kinase genes occurs early during spermatogenesis, but high level transcription detectable by Northern analysis occurs during spermiogenesis.

In agreement with this interpretation, very low (basal) levels of transcripts of the T66Bk-rsk3 fusion gene are detectable by RT-PCR at stage 7, 14 and 20 days p.p., but much higher levels can be seen only from stage 22 d.p.p. onwards (FIG. 3a). This suggests that the T66Bk-rsk3 fusion gene is up-regulated at about the stage when elongating spermatids appear (see below).

The genes mouse protamine 1 (mP1), angiotensin converting enzyme (ACE) and c-kit were analyzed in order to allow a staging of the onset of the T66Bk-rsk3 fusion gene expression during spermatogenesis (FIG. 3b). mP1 has been reported to be first expressed in round spermatids (Peschon et al. 1987), the testis specific promoters of ACE (Howard et al. 1990) and c-kit (Rossi et al. 1992) are first active in elongating spermatids of undefined stage and stage IX-XI, respectively. The analysis of all three promoters has been achieved using transgenic animals (Langford et al. 1991; Albanesi et al. 1996; Peschon et al. 1987). In the RT-PCR analysis shown here, mP1 transcripts were detected as early as day 14 p.p., but a strong band appeared at day 18 p.p. According to Rugh (1990), spermatids appear at day 17 p.p. in male pups. The ACE and c-kit testis transcripts were weakly detectable at 20 days p.p., but a signal comparable to the T66Bk-rsk3 fusion gene band 181/144 first appeared at 22 days p.p. An earlier expression of ACE was detected in day 7 and 14 p.p. testis. Thus, the RT-PCR data are in agreement with the published data showing that ACE and c-kit are expressed in elongating spermatids. This suggests that the expression of the T66Bk-rsk3 fusion gene in testis is up-regulated at about the same time or a little later than that of c-kit and ACE, in elongating spermatids, and that the promoter of the T66Bk-rsk3 fusion gene may be active late enough during spermiogenesis to exclude the distribution of the T66Bk-rsk3 fusion gene products to spermatocytes not containing the T66Bk-rsk3 fusion gene (Willison and Ashworth 1987), thus fulfilling an important criterion for the R function. The low level of expression found in day 7 and 14 p.p., but not in day 18 p.p. testis suggests that the transcripts might be degraded by the end of meiosis.

EXAMLPE 4 T66B-related Genes are Conserved During Evolution

Putative homologs of the T66Bk-related kinases also exist in other species (FIG. 6). A Southern blot hybridization assay at reduced stringency using the cDNA fragment 191/144 as a probe revealed cross-hybridizing fragments in hamster, rabbit, pig, chick and human. This suggests a conservation of the T66Bk-related kinases in other mammals as well as in birds.

EXAMLPE 5 The Mouse T/t-complex Encodes Several Members of the T66Bk Gene Family

In a Southern blot hybridization of cosmid cat.15 with the probe pCRt^(h2)-161/170 three hybridizing BamHI fragments, B7.8, B9.1 and a 6.1 kb BamHI fragment are detected (see FIG. 2). Sequencing of the T66Bk or related gene encoding parts of these genomic DNA fragments revealed that each of the BamHI fragments B7.8 and B9.1 contains a large open reading frame (ORF) encoding T66Bk and another member of the T66Bk gene family, respectively. The centromere farthest BamHI fragment (B7.8) contains the T66Bk ORF (FIGS. 1 and 4a and b). Its transcribed part (exon) differs from the corresponding exon contained in the cDNA pCRt^(h2)-161/144 by a single point mutation (base 1490 C to T) probably due to an allelic variation between the t-haplotypes t^(h2), and t^(w12) from which cosmid cat.15 was derived, resulting in a single amino acid exchange (Pro to Leu).

The next centromere closer BamHI fragment (B9.1) contains 5′-noncoding sequence and most likely the promoter of T66Bk and, further upstream of it, an ORF encoding exon and a 3′-noncoding exon of another member of the T66Bk gene family, named here T66Bk-2. However, in this case the 3′-noncoding exon is not related to rsk3. The exon sequence of T66Bk-2 encoding a large ORF is shown on FIGS. 7a and b. It differs from the ORF of T66Bk in a number of positions; nevertheless, it is very closely related to T66Bk. In the t⁶/+ testis cDNA panel, expression of T66Bk-2 is first detected at 22 days p.p. Considerably higher expression is observed from 24 days p.p. onwards (FIG. 8).

The mouse genome contains several more loci of the T66Bk gene family some of which are located in the region of the T/t-complex distal to T66B, probably in T66C. This is based on the observation of several BamHI fragments hybridizing to pCRt^(h2)-161/170, other than those described above, contained in the genome of mice carrying partial t-haplotypes or wild type mice. Some of these BamHI fragments are polymorphic and specific to complete t-haplotypes, but are not present in the partial t-haplotypes t^(h44), t^(Jr1), t^(lowH), t^(h2) or t⁴⁹ nor in wild type (see FIG. 2). Therefore they must be contained in the T/t-complex region distal to T66B. To obtain coding sequences of T66Bk gene family members not contained in the T66B region several cDNA clones were isolated from a testis cDNA library constructed from male mice of the genotype t^(w5)/t^(w12), by hybridization with the probe pCRt^(w5)-155/170 derived from the T66Bk gene. Several cDNA clones were isolated. All of them have a high sequence similarity to T66Bk or T66Bk-2.

One of them, T66k-8 (FIGS. 7c, d, and e) is almost identical in sequence to T66Bk-2 as far as sequence is available for both genes, except that it contains a single base deletion leading to an alteration of the ORF C-terminally to the protein kinase domain. From the high sequence conservation of T66k-8 to T66Bk-2 it seems not unlikely that T66k-8 is derived from the T66Bk-2 locus. However, it is not clear how the single base change was introduced into the cDNA clone, whether by a mistake in the RNA transcription, processing, reverse transcription, or by another mechanism. For instance, it has been shown that RNA editing resulting in a change of the nucleotide sequence which can alter the ORF, can occur in lower and higher eukaryotes. At the moment, such a mechanism cannot be excluded as the cause of the observed alteration. Nor can it be excluded that T66k-8 derives from a duplicated T66Bk-2 locus. Alternatively, T66k-8 might be derived from the t^(w5) allele of T66Bk-2. Another cDNA was found that also contains a single base deletion at a similar position as T66k-8. The genomic location of the corresponding gene has not been determined. The alteration predicted for the C-terminal tail of either gene product would be expected to result in a change of the regulation and/or level of their protein kinase activity and/or of the location of the protein within the cell.

Another cDNA clone, T66k-7as (FIGS. 7f, g, and h), also isolated from the cDNA library, has a very intriguing sequence and structure. It contains a sequence strongly related to T66BkfT66Bk-2, including intron sequences from either side of the exon containing the single long ORF and additional sequences from further downstream, inserted in antisense orientation in the plasmid cDNA vector. Therefore T66k-7as must be derived from an antisense transcript of a T66Bk family gene. The predicted T66k-7as transcript does not contain a long ORF. The intron sequence 5′ to the ORF encoding exon of T66Bk/T66Bk-2 is very A/T rich in antisense direction and apparently serves as transcription stop and polyadenylation signal during the synthesis of this antisense transcript. The sequences contained in the BamHI fragment B9.1 of cat.15 which are related by sequence to the 5′ end of T66k-7as map to the vicinity of the promoter of T66Bk suggesting that the promoter region of T66Bk might contain elements controlling in cis the transcription of T66Bk sense RNA as well as the transcription of T66Bk-2 antisense RNA. If that were the case, antisense transcription might be achieved by the same cis-control elements and thus occur at the same stage as sense-RNA transcription. So far, no antisense transcript coming from that locus of the T66B region was identified. Nonetheless, the similarity of the structure and sequence of T66Bk-7as to the head-to-tail arrangement and sequence of T66Bk-2fT66Bk suggests that the T66Bk-2 gene of the T66B region might be transcribed in antisense direction. In addition, another T66Bk locus must exist which is transcribed in antisense direction, gave rise to the cDNA T66k-7as and might be located within the T66B region.

It is obvious that the expression of antisense transcript complementary to mRNA transcribed from members of the T66Bk gene family would be well suited to diminish the level of functional gene products derived from that gene family. This could influence the spermatozoa in two ways. If the antisense transcripts act in both types of spermatids, those carrying the t-Responder and those not carrying it, the former might be protected from that negative action of antisense transcripts by a higher activity of its T66Bk family gene products whereas the latter are not. In the alternative, more likely way the antisense RNA transcripts might be restricted to the former spermatids and lower the expression of T66Bk gene products expressed in them. This would help to protect the former from the negative action of hypermorphic Distorter gene products, whereas the latter would be “poisoned” by them. This “poisoning” would be caused by hyperactivation of the Responder/Distorter signaling cascade.

Antisense RNA derived from (a) T66Bk family member(s) would be expected to attenuate the negative effect of the Distorters and, in that way is envisaged to contribute to the transmission ratio distortion phenotype. Another cDNA clone, T66k-20, isolated from the t^(w5)/t^(w12) testis cDNA library encodes yet another member of the T66Bk gene family (FIGS. 7i, j, and k). Its ORF differs from T66Bk and T66Bk-2 in a number of amino acid residues and in particular at the N-terminal end which is 20 residues longer than that of T66Bk and T66Bk-2 (FIG. 7e). Most likely, T66k-20 is derived from a gene located in the T66A region, and thus may provide wild type Responder activity.

The analysis of the transmission ratios of t^(lowH) or t^(low3H) heterozygous with t^(h51)t^(h18) by Lyon (1984), showed a strong difference between the transmission ratio of t^(lowH) and t^(low3H). In addition, neither t-haplotype reached the high value of a complete t-haplotype heterozygous with a wild type chromosome. These data suggest the involvement of several loci in the t-Responder function. At the present level of analysis it is speculated that T66Bk, T66Bk-2, T66k-8, T66k-20 and T66k-7as may cooperatively contribute to the t-Responder function.

The testis cDNA library prepared from RNA of a male carrying the t-haplotypes t^(w5)/t^(w12) did not contain a cDNA clone derived from the T66Bk gene. Therefore another testis cDNA library was constructed from RNA of a male carrying the t-haplotypes t⁶/t^(w5). Four clones containing a fragment of the size expected from PCR amplification with the primer pair 161/144 were identified and one of them was purified and sequenced (FIG. 9). The sequence is identical to that of the cDNA pCRt^(h2)-161/144 (FIGS. 4a and b) in the region of overlap and extends it at the 5′ as well as the 3′-end. It is worth noting that the sequence ends in an intron of the rsk3 locus in the T66B region and has no consensus polyadenylation signal suggesting that the cDNA is not derived from a properly processed mRNA molecule, but from a, possibly rare, transcript which has not been spliced completely and may contain a dA-rich intron sequence. This finding leaves open the possibility that the T66Bk gene transcript might include the complete rsk3 locus in T66B from bp 438 of the coding region to the 3′-end.

In addition to the T66Bk family members encoded in the t-haplotype, three more family members derived from the wild type inbred strains Balb/c, C3H/N and 129/Sv were isolated either by RT-PCR or on a genomic clone (FIG. 10). Again, high sequence conservation to the t-haplotype family members was observed. The gene pCR.Balb-66k has the same feature as the gene T66k-20, namely a potential translation start site upstream of the one utilized by T66Bk coding for additional 20 amino acid residues. It is not clear, however, whether this translation start is efficiently used since it does not conform with Kozak's rules demanding an A or a G at position-3 upstream of the ATG codon.

In contrast, the genes pCR.C3H-66k and pλ.129-66k differ significantly from all other T66Bk family members at their C-terminus. Both genes contain a translation stop codon at triplet position 434 resulting in a truncated protein of only 433 amino acid residues whereas the remaining nucleic acid sequence is not significantly different from those of the other members. The truncation occurs outside the kinase domain suggesting that the protein might still be able to function as a kinase. However, the alteration of the C-terminus might influence the regulation and/or level of kinase activity. In this context it is interesting to note that on the C3H background t-haplotypes are transmitted at a very high ratio, whereas e.g. t^(o) is transmitted at a reduced ratio from males carrying the T/t-complex from Balb/c compared to the ratio obtained by males of the genotype t^(o)/C3H (Bennett et al. 1983). The 129Sv background also enhances the transmission ratio of t-haplotypes similar to C3H (our observations). The shortened ORFs in pCR.C3H-66k and pλ.129-66k might have an influence on this behaviour. On the other hand, other T66Bk family members encoding proteins of the same length as T66Bk might exist in these strains in addition to the ones shown here.

Therefore, and in general, it is to be noted that the genetic background of the animal strain involved may significantly contribute to the expression of the phenotype in terms of the level of distortion of the transmission ratio.

EXAMLPE 6 Transmission Ratio Distortion in Males Carrying Transgene Insertions Encoding the T66Bk Kinase

To prove the involvement of T66Bk in the Responder phenotype transgene constructs were made expressing the kinase gene T66Bk (FIGS. 4a and b) either under control of the testis promoter of c-kit (tg4-3; tg4-13) or of the putative endogenous promoter of T66Bk (FIG. 11) in transgenic mice (tg5-43; tg5-25). Mice carrying the trangene integration were mated to mice carrying either the t-haplotype t^(h51)−t^(h18) expressing the t-Distorters D1 and D2 or the wild type chromosomes C57BL/6 or Ttf/+tf (Lyon 1984). Males of the appropriate genotype were mated to NMRI outbred females and their offspring tested for carriers of the transgene. The expectation based on the experiments of Lyon (1984) was that, if T66Bk encodes a protein involved in transmission ratio distortion the t-Distorters should enhance the transmission ratio of the transgene, as is the case in the genotype +t^(lowH)+/t^(h51)+t^(h18), whereas in males carrying wild type chromosomes the transmission ratio of the transgene should be lowered. Table 1 shows the data obtained so far. Interestingly, one of the transgene integrations (tg4-3) must have occurred on the Y chromosome since it is only observed in males. In this case offspring were examined for external sexual characteristics after birth, the other transgene integrations were examined by PCR analysis. The data demonstrate a significant distortion of the transmission of the transgene confirming that T66Bk encodes t-Responder activity. The data also demonstrate the potential of the T66Bk gene in breeding strategies selecting for specific genetic traits, in particular sex. In addition the data show the usefulness of both promoters as control elements in achieving a Responder phenotype.

However, the transmission distortion effect obtained is considerably smaller than that observed with the genotype +t^(lowH)+/t^(h51)+t^(h18) or +t^(lowH)+/++tf (Lyon 1984). This suggests that either the expression level of the T66Bk kinase from the transgene constructs is not adequate or that the expression of wild type Responder loci in spermatozoa carrying the transgene diminishes the effect of the T66Bk gene. It should be taken into consideration that the t^(lowH) chromosome is carrying loci selected by nature for an optimal effect on transmission ratio distortion. In Lyon's analyses (1984) sperm carrying this chromosome compete with sperm carrying either a wild type chromosome or the t-Distorters t^(h51)−t^(h18) probably in combination with (a) wild type Responder locus (loci). In contrast, the trangene integrations occurred outside of chromosome 17. Therefore, transgene expression always occurs in sperm expressing in addition (a) wild type Responder locus (loci). These sperm are competing with sperm carrying either a wild type chromosome or the t-Distorters t^(h51)−t^(h18) probably in combination with (a) wild type Responder locus (loci). The combination of T66Bk expressed from the transgene with expression products from (a) wild type Responder locus (loci) might be less effective in distorting the transmission ratio than the combination of products expressed by members of the T66Bk gene family, in particular T66Bk and T66Bk-2, in the t^(lowH) t-haplotype. Also, it has been demonstrated that the genetic background has a considerable effect on the ratio of transmission distortion achieved by various t-haplotypes (Bennett et al., 1983). It is quite clear that the expression level and/or activity of the T66Bk gene has to be optimized in future experiments in order to obtain a stronger transmission ratio distortion effect.

Also, control elements affecting the expression level such as elements regulating transcription efficiency, transcript processing and stability and translation efficiency, used for transgene expression have to be optimised to achieve a maximal effect. It would be convenient to select a tissue and stage specific promoter such as the one. controlling the expression of T66k-20 preferably including its 5′-untranslated region, first intron and 3′-untranslated region. Alternatively, an 3′-untranslated region known to increase the stability of the corresponding mRNA could be used. We have noticed that transcripts derived from T66k-20 are respresented at a high ratio in cDNA isolated from a testis cDNA library constructed from RNA of mice carrying t^(w5)/t^(w12). In contrast, cDNAs derived from T66Bk were not found and cDNAs derived from T66Bk-2 were highly underrepresented, suggesting that the transcription level of T66k-20 is considerably higher than that of the former loci.

However, transfer of this system for distortion of the transmission of genetic traits, in particular of sex, to farm animals might be achievable without a major effort since it is not expected that amplification of T66Bk related genes also occurred in farm animals which have not evolved transmission ratio distortion. Therefore, T66Bk might have a much stronger effect on transmission ratio when introduced into farm animals. The data presented here open the prospect of producing farm animals fathering preferentially or even exclusively offspring of the same sex, e.g. only or predominantly females.

EXAMLPE 7 Cloning of Wild Type Members of the T66Bk Kinase Gene Family

The cDNAs pCR.Balb-66k and pCR.C3H-66k were isolated by RT-PCR using the primer pairs 161/220 (220: 5′-CTTCCCCCTGGCTGGAC-3′ (SEQ ID NO:24)) from testis RNA of the inbred strain Balb/c and C3H/N, respectively, cloned in the plasmid vector pCR2.1 (Invitrogen) and analyzed using the methods described in figure legends 3 and 4. The extension step in the PCR was performed for 2 min. at 72° C. The sequence of p_(—).129-66k was derived from an EcoRI subclone in pBluescriptKS made from a lambda-FixII clone isolated from a genomic lambda-FixII library using a cDNA fragment of T66Bk as probe. The lambda-library was constructed from genomic DNA of the ES-cell line R1 (Nagy et al. 1993), according to the instructions of the supplier for the lambda cloning and packaging kits (Stratagene). Library construction, plating and screening by hybridization was according to standard techniques (Sambrook et al. 1989) and the methods described in figure legends 2, 3 and 4.

Primer Sequences:

ACE

5′GC CAA CCA GGG GAT A 3′(SEQ ID NO:25); 5′CT GTC CGG TCA TAC TCT T 3′(SEQ ID NO:26)

c-kit

5′CTT GTG TCC TTG GGA GAA 3′(SEQ ID NO:27); 5′GGT GCC ATC CAC TTC AC 3′(SEQ ID NO:28)

mP1

5′CGC AGC AAA AGC AGG AGC AG 3′(SEQ ID NO:29); 5′CAT CGG ACG GTG GCA TTT TT 3′(SEQ ID NO:30)

mouse rsk3

144: 5′TGC TCA AGC CAA AAT CTG TG 3′(SEQ ID NO:31)

145: 5′ATG GCC TGG GGA TCA TCT AC 3′(SEQ ID NO:32)

146: 5′CAC CGC TTG CAC ACT GAG TA 3′(SEQ ID NO:33)

cDNA pCRt^(h2)-161/144

155: 5′ATC GAT GTG TGG GGT CTT 3′(SEQ ID NO:34)

161: 5′GTT TGG GAG GAG CTT GTG 3′(SEQ ID NO:35)

170: 5′CTA GTC CAG CCC TTG ATG 3′(SEQ ID NO:36)

181: 5′TGG CAT CTT ATT GTC TAC 3′(SEQ ID NO:37)

191: 5′CCA AGC CCC UT TTC TGA 3′(SEQ ID NO:38)

pSV-Sport1

seq5lib: 5′ATTTAGGTGACACTATAGAAGGTA 3′(SEQ ID NO:39)

Oligonucleotide Sequences:

232: 5′CCC CCT TTA TCT GAC 3′(SEQ ID NO:40)

237: 5′TAT GCT GGC AGC ATC AAA 3′(SEQ ID NO:41)

TABLE 1 tg4 males genotype # female # male % male 4-3/5 th51-th18/C57BL 42 71 62.8 4-3/36 th51-th18/C57BL 33 55 62.5 4-3/39 th51-th18/C57BL 50 67 57.2 total: 125 193 60.7% 4-3/37 +tf/C57BL 42 29 40.8 4-3/187 C57BL/C57BL 52 37 41.6 total: 94 66 41.2% tg4 males genotype # −tg # +tg % tg 4-13/80 th51-th18/C57BL 41 58 58.6 4-13/86 th51-th18/C57BL 45 55 55 4-13/97 th51-th18/C57BL 44 56 56 total: 130 169 56.5% 4-13/53 +tf/C57BL 56 47 45.6 4-13/96 +tf/C57BL 70 67 48.9 4-13/100 +tf/C57BL 53 47 47 total: 179 161 47.3% tg5 males genotype # −tg # +tg % tg 5-43/100 th51-th18/C57BL 13 29 69.0 5-43/101 th51-th18/C57BL 12 16 57.1 5-43/104 th51-th18/C57BL 26 28 51.8 5-43/105 th51-th18/C57BL 12 25 67.5 total: 63 98 60.8% 5-25/83 Ttf/C57BL 43 29 40.3 5-25/84 +tf/C57BL 37 24 39.3 total: 80 53 39.8%

Table 1

Transmission ratio distortion in mice carrying transgenes encoding the kinase gene T66Bk.

Two transgene constructs, tg4 and tg5 containing the protein coding region of T66Bk were constructed in vitro and introduced into the germ line by injection of DNA into one pronucleus of fertilized eggs of the genotype ((C57BU6 ×C3H/N)F1 ×C57BU6) female ×NMRI male and retransfer of the zygotes or 2-cell embryos into NMRI foster mothers. Male or female carriers of either transgene were mated to mice carrying either the t-Distorters D1 and D2 on a single t-haplotype chromosome (t^(h51)−t^(h18)) over Ttf, +tf or C57BL/6, or either the wild type genotype Ttf/+tf or C57BL/C57BL. Males carrying the appropriate genotype were identified by PCR analysis and set up for test matings with NMRI outbred females. In most cases, late embryonic stages were used as source of DNA for testing individual offspring for the presence or absence of the transgene, the remainder were tested using a tail piece as DNA source. A chromosome 17 marker locus was tested in parallel to control the quality of the DNA solution. The transgene tg4 of the line 4-3 segregates with the Y-chromosome, suggesting that tg4 is integrated on the Y chromosome. Therefore, in this case, offspring were examined after birth for their sex using external sexual characteristics. The breeding data demonstrate non-mendelian inheritance of the transgene and, in the case of tg4-3, of sex. The deviation from the expected 50% depends on the presence or absence of t-Distorter loci, being significantly higher than 50% in the presence and lower than 50% in the absence of t-Distorter loci, as expected from the t-haplotype Responder locus Tcr. This confirms the finding that T66Bk encodes t-Responder activity.

Methods:

Tg4 consists of the testis promoter of c-kit, base 45 to the Styl site at base 683 (Rossi et al. 1992; Albanesi et al. 1996), the cDNA t^(h2)-161/144 and additional mouse rsk3 sequence comprising bp 438 up to bp 998 of rsk3 (Kispert, 1990), and IRES-βgeo containing the internal ribosome entry site IRES (Ghattas et al. 1991) and the βgal-neo fusion gene and SV40 polyadenylation signal (Friedrich and Soriano 1991). In brief, the testis promoter of c-kit was isolated by RT-PCR from testis RNA using the primer pair 5′-ATGTAAGTGGCATGGAGT-3′ (SEQ ID NO:42) and 5′-GCACACCGAAAATAAAA-3′ (SEQ ID NO:43) and cloned into the plasmid vector pCR2.1 (Invitrogen). A Notl-BstEII fragment comprising the cDNA t^(h2)-161/144 from a vector NotI site at the 5′-end to a BstEII site in the rsk3 homology region was ligated to Notl and BstEII sites in the plasmid IRES-βgeo containing the rsk3 homology region from the BstEII site to bp 998, 5′ of the IRES-βgeo gene. The 5′-end of the resulting construct containing an EcoRV site from the vector pCR2.1 just 3′ of the NotI site was replaced by a NotI-StyI fragment containing the testis promoter of c-kit cloned in the vector pCR2.1 by ligation of the NotI-StyI(blunt; the Styl site was blunt-ended by treatment with the Klenow-fragment of E.coli DNA polymerase I) fragment comprising bp 45 to bp 683 of the c-kit promoter into the NotI and EcoRV sites of the construct. The final transgene construct was released from the vector by digestion with Notl and SaII.

Tg5 consists of 2637 bp (KpnI to PmII fragment) of the genomic region upstream of the putative transcription start site of T66Bk including most of the 5′-untranslated region and the putative promoter of T66Bk (FIG. 11), the cDNA t^(h2)-161/144 from the HincII site (bp 293) to the EcoRI site in vector pCR2.1 including the complete protein coding region and a HA-tag constructed into the start site of translation, the IRES sequence and coding region of human CD24 (Kay et al. 1991), and the modified intron and polyadenylation signal of SV40-t (Huang and Gorman 1990). Tg5 was constructed in several steps. First, an HA-tag encoding the peptide sequence YPYDVPDYA was introduced at the translation start of the cDNA t^(h2)-161/144.

Second, the putative promoter of T66Bk was isolated as a 2.6 kb KpnI(blunt)-PmII fragment from the genomic BamHI fragment B9.1 of cosmid cat.15, and ligated into EcoRV and HincII sites of the vector containing the HA-tagged cDNA t^(h2)-161/144. The EcoRV site stems from the vector pCR2.1 while the HincII site is contained in the 5′-untranslated region of the cDNA t^(h2)-161/144. In the third step the IRES sequence and hCD24 coding sequence was cut as an EcoRI-EagI(blunted) fragment from the plasmid pSLV-1, the modified intron and polyadenylation signal of SV40-t were cut as a SnaBI-BamHI fragment from the Vector pSV-Sport1 (Gibco/BRL), and both fragments were ligated together into the previous construct opened at the vector sites EcoRI and BamHI located at the 3′-end of the insert. The construction of an HA-tag into the translation start site of T66Bk was done as follows. First, two fragments of the cDNA t^(h2)-161/144 were amplified by PCR using the primer 5′-GGCGTAGTCTGGGACGTCGTATGGGTACATGTCAGAAAAAGG-3′ (SEQ ID NO:44) and 5′-ATGTACCCATACGACGTCCCAGACTACGCCATGGAGAAATTTCAT-3′ (SEQ ID NO:45), respectively, in combination with the upstream primer 161 or the downstream primer 188 (5′-ACCCTGGTTGTGGCAGTA-3′(SEQ ID NO:46)), respectively, creating an overlapping region encoding the HA-tag sequence coding for the peptide YPYDVPDYA (SEQ ID NO:53), in frame with the translation start site of T66Bk. The PCR was performed as described in figure legend 3 except that 15 cycles were performed and 50 ng template were added. Then, both fragments were isolated from an agarose gel and used as template together in a second PCR. First 15 cycles of 30 sec. 94° C., 2 min. 72° C. were performed without primers, the flanking primers 161 and 188 were added and a further 25 cycles of 30 sec. 94° C., 30 sec. 50° C., 30 sec. 72° C. were performed. The resulting fragment containing the HA-tag sequence was purified from an agarose gel, cut with HincII-EcoNI and ligated in place of the HincII-EcoNI fragment of the original cDNA clone t^(h2)-161/144.

Testing of offspring for carriers of the transgene insertion was done by first digesting a tissue sample of individual embryos or mice in lysis buffer (100 mM Tris-HCI pH8.5/5 mM EDTA/0.2% SDS/200 mM NaCl/200 μg/ml Proteinase K) over night at 55° C., diluting an aliquot 20 fold in water followed by inactivation of the Proteinase K by incubation at 80° C. for 30 min., and assaying 1 μl in a 20 μl PCR reaction as described in figure legend 3 using the primer pair 309: 5′-CAGCCCATGAATCCATC-3′ (SEQ ID NO:47) and 310: 5′-TGCCTTCGGTCTGAAAG-3′ (SEQ ID NO:48) and the cycling conditions 2 min. 94° C., 35 cycles 30 sec. 94° C., 30 sec. 50° C., 1 min. 72° C. A control PCR reaction assaying for the genotype at the locus Hba-4ps in the distal region of the mouse T/t-complex was performed where appropriate using the primer pair Hb.1/Hb.2 and conditions as published (Schimenti and Hammer 1990). This PCR reaction was also used to test for the presence of the distal t-haplotype region t^(h18) containing the t-Distorter D2. Likewise, presence of the proximal t-Distorter D1 in the t-haplotype t^(h51) was assayed by testing for the presence of a t-specific fragment at the Tcp1 locus. This was done by PCR using the primer pair 5′-AGGAAAGCTTGCCCMGAGAATAGTTMTGC-3′ (SEQ ID NO:49) and 5′-AGGCGAATTCCATATCATCAATGCCACCAG-3′ (SEQ ID NO:50). The cycling conditions were 40 sec. 94° C., 40 sec. 60° C., 1 min. 30 sec. 72° C., 35 cycles. Different wild type alleles at the locus D17Mit46 from the middle of the T/t-complex were distinguished by PCR using the primers Left: 5′-TCCACCCCACTACCTGACTC-3′ (SEQ ID NO:51) and Right: 5′-CCCTTCTGATGACCACAGGT-3′ (SEQ ID NO:52). Cycling conditions were 40 sec. 94° C., 40 sec. 50° C., 40 sec. 72° C., 35 cycles. This marker allows to distinguish between the allelic variants of the strains C57BU6, NMRI and Ttf/+tf.

All cloning procedures were performed according to standard techniques (Sambrook et al. 1989), the production of transgenic mice was done according to the methods described in Methods in Enzymology, Vol. 225, Guides to Techniques in Mouse Development, 1993 (ed. P. M. Wassarman and M. L. DePamphilis). Mice carrying the t-haplotype t^(h51)−t^(h18) were obtained from Dr. M. F. Lyon (Harwell, England), mice with the genotype Ttf/+tf were a gift of Dr. K. Artzt (Austin, Tex.).

References

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53 1 2076 DNA Mus musculus CDS (337)..(1788) 1 gtttgggagg agcttgtgtg tgtgagttgt gttttaagtt tatttgcgtg tgagtacctt 60 tgggtttttg tgtgtgtctg tgtgtgtttg tgtgtgtata actgtgggtg actgtaagtg 120 cacctgtgtg tttgtacgtg agtgtgtaag actgtgtgtg tgcacaagag cgtgtgtagg 180 tgcacgtgtt gtaggtgtga gaacacctgt tgtgtttagg ccatcagtca gcttggtcat 240 tgtttctaag gtagcattta tactttgtta cctcaagtgg gctctgggag tcaacagaag 300 tcagaaaagc tcagatccaa gccccctttt tctgac atg gag aaa ttt cat gct 354 Met Glu Lys Phe His Ala 1 5 caa tat gag atg cta gag act att ggc cag gga ggc tgc gcc cag gtg 402 Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln Gly Gly Cys Ala Gln Val 10 15 20 aag ctg gcc cga cac cgc ctc aca ggc acc cac gtg gct gtc aaa gtg 450 Lys Leu Ala Arg His Arg Leu Thr Gly Thr His Val Ala Val Lys Val 25 30 35 att gta aag agg gag tgt tgg ttc aac cct gtc atg tct gag gca gag 498 Ile Val Lys Arg Glu Cys Trp Phe Asn Pro Val Met Ser Glu Ala Glu 40 45 50 tta ctg atg atg acc gat cat ccg aat atc atc tct ctc ctt caa gtc 546 Leu Leu Met Met Thr Asp His Pro Asn Ile Ile Ser Leu Leu Gln Val 55 60 65 70 att gag acc aag aag aaa gta tac ctc att atg gag ttg tgc gag ggt 594 Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile Met Glu Leu Cys Glu Gly 75 80 85 aaa tca ctt tac caa cac atc caa aat gct ggc tac ctg cag gag gat 642 Lys Ser Leu Tyr Gln His Ile Gln Asn Ala Gly Tyr Leu Gln Glu Asp 90 95 100 gaa gca cgc cca tta ttc aag cag ctc tta agt gct atg aac tac tgc 690 Glu Ala Arg Pro Leu Phe Lys Gln Leu Leu Ser Ala Met Asn Tyr Cys 105 110 115 cac aac cag ggt ata gtt cac agg gac ctg aca cct gac aat att atg 738 His Asn Gln Gly Ile Val His Arg Asp Leu Thr Pro Asp Asn Ile Met 120 125 130 gta gaa aaa gat ggg aaa gtg aag atc att gat ttt gga ctc ggc acc 786 Val Glu Lys Asp Gly Lys Val Lys Ile Ile Asp Phe Gly Leu Gly Thr 135 140 145 150 caa gag aag cca ggg caa aac cac aac tta ttc tgt gag att tac cca 834 Gln Glu Lys Pro Gly Gln Asn His Asn Leu Phe Cys Glu Ile Tyr Pro 155 160 165 ttt agt act cct gag gtg ctc ttt aac aga ccc tat gat atg cgc aag 882 Phe Ser Thr Pro Glu Val Leu Phe Asn Arg Pro Tyr Asp Met Arg Lys 170 175 180 atc gat gtg tgg ggt ctt gga gtt gtg ctg tat ttt atg gta act gga 930 Ile Asp Val Trp Gly Leu Gly Val Val Leu Tyr Phe Met Val Thr Gly 185 190 195 aag att ctg ttt gat act gcc agc gta gaa aag ctg cga aag caa att 978 Lys Ile Leu Phe Asp Thr Ala Ser Val Glu Lys Leu Arg Lys Gln Ile 200 205 210 gtt gca gaa aag tgt tct gtt ccc tgt aga ctg tca gta gag ctc caa 1026 Val Ala Glu Lys Cys Ser Val Pro Cys Arg Leu Ser Val Glu Leu Gln 215 220 225 230 gac ctg att aga ctt tta atg acg gac atc ccc gaa ctt agg ccc act 1074 Asp Leu Ile Arg Leu Leu Met Thr Asp Ile Pro Glu Leu Arg Pro Thr 235 240 245 gtt gct gaa gtt atg gtg cat ccc tgg gtc aca gaa ggc tca ggg gtg 1122 Val Ala Glu Val Met Val His Pro Trp Val Thr Glu Gly Ser Gly Val 250 255 260 tta cca gat cct tgt gaa gaa cat ata ccc ctc aag cca gac cct gcg 1170 Leu Pro Asp Pro Cys Glu Glu His Ile Pro Leu Lys Pro Asp Pro Ala 265 270 275 att gca aaa gca atg gga ttt atc ggg ttc caa gct caa gac att gaa 1218 Ile Ala Lys Ala Met Gly Phe Ile Gly Phe Gln Ala Gln Asp Ile Glu 280 285 290 gat tcg tta tgt cag aga aaa ttc aac gaa acc atg gca tct tat tgt 1266 Asp Ser Leu Cys Gln Arg Lys Phe Asn Glu Thr Met Ala Ser Tyr Cys 295 300 305 310 cta ctg aaa aaa cag att ctt aag gaa tgt gac agg cca atc cgg gct 1314 Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys Asp Arg Pro Ile Arg Ala 315 320 325 cag ccc atg aat cca tct gtg acc cca ctc tct tcc ctt gtt gat gct 1362 Gln Pro Met Asn Pro Ser Val Thr Pro Leu Ser Ser Leu Val Asp Ala 330 335 340 cct act ttc cat ctc gga ctt cgg agg aca gag act gaa ccc aca ggt 1410 Pro Thr Phe His Leu Gly Leu Arg Arg Thr Glu Thr Glu Pro Thr Gly 345 350 355 ctc aga tta tct gac aat aag gaa gtg cct gtc tgt ggc aat agt act 1458 Leu Arg Leu Ser Asp Asn Lys Glu Val Pro Val Cys Gly Asn Ser Thr 360 365 370 agt aag aaa aga gag aga agt ttc agt ggg ccg ggt gtt ctc agc agg 1506 Ser Lys Lys Arg Glu Arg Ser Phe Ser Gly Pro Gly Val Leu Ser Arg 375 380 385 390 ccg att aac aca aca ccc aca atg gac caa aca cac acc cgt act tgg 1554 Pro Ile Asn Thr Thr Pro Thr Met Asp Gln Thr His Thr Arg Thr Trp 395 400 405 agt ggt ccc tgc att tac tca aat gtt tgc aca atc cat cca aac agc 1602 Ser Gly Pro Cys Ile Tyr Ser Asn Val Cys Thr Ile His Pro Asn Ser 410 415 420 atc aat gag agt aca gaa ggc cac atc agt acc tca gca gag gat aag 1650 Ile Asn Glu Ser Thr Glu Gly His Ile Ser Thr Ser Ala Glu Asp Lys 425 430 435 cct gtc cac agc aga ggc tgg ccc aga ggc atc aag ggc tgg act agg 1698 Pro Val His Ser Arg Gly Trp Pro Arg Gly Ile Lys Gly Trp Thr Arg 440 445 450 aag ata gga aat gca atg agg aag ctc tgt tgc tgt atc cca tcc aaa 1746 Lys Ile Gly Asn Ala Met Arg Lys Leu Cys Cys Cys Ile Pro Ser Lys 455 460 465 470 gag aca tct cac ctg ggg cag aga aga gtc tgc cca aaa att 1788 Glu Thr Ser His Leu Gly Gln Arg Arg Val Cys Pro Lys Ile 475 480 taagacacag gaaggatgtc aggagaatga gcatccagca tggcccagcc tttcagaccg 1848 aaggcaagct ctacctgatc ctggacttcc tgcggggagg tgacctcttc accaggcttt 1908 ccaaagaggt gatgttcacg gaggaggatg tcaagttcta cctggctgag ctggccttgg 1968 ctctagacca cctccatggc ctggggatca tctacaggga tctgaagcca gagaatatcc 2028 tcctggatga agagggacat attaagatca cagattttgg cttgagca 2076 2 484 PRT Mus musculus 2 Met Glu Lys Phe His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln 1 5 10 15 Gly Gly Cys Ala Gln Val Lys Leu Ala Arg His Arg Leu Thr Gly Thr 20 25 30 His Val Ala Val Lys Val Ile Val Lys Arg Glu Cys Trp Phe Asn Pro 35 40 45 Val Met Ser Glu Ala Glu Leu Leu Met Met Thr Asp His Pro Asn Ile 50 55 60 Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile 65 70 75 80 Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala 85 90 95 Gly Tyr Leu Gln Glu Asp Glu Ala Arg Pro Leu Phe Lys Gln Leu Leu 100 105 110 Ser Ala Met Asn Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu 115 120 125 Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Lys Val Lys Ile Ile 130 135 140 Asp Phe Gly Leu Gly Thr Gln Glu Lys Pro Gly Gln Asn His Asn Leu 145 150 155 160 Phe Cys Glu Ile Tyr Pro Phe Ser Thr Pro Glu Val Leu Phe Asn Arg 165 170 175 Pro Tyr Asp Met Arg Lys Ile Asp Val Trp Gly Leu Gly Val Val Leu 180 185 190 Tyr Phe Met Val Thr Gly Lys Ile Leu Phe Asp Thr Ala Ser Val Glu 195 200 205 Lys Leu Arg Lys Gln Ile Val Ala Glu Lys Cys Ser Val Pro Cys Arg 210 215 220 Leu Ser Val Glu Leu Gln Asp Leu Ile Arg Leu Leu Met Thr Asp Ile 225 230 235 240 Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val His Pro Trp Val 245 250 255 Thr Glu Gly Ser Gly Val Leu Pro Asp Pro Cys Glu Glu His Ile Pro 260 265 270 Leu Lys Pro Asp Pro Ala Ile Ala Lys Ala Met Gly Phe Ile Gly Phe 275 280 285 Gln Ala Gln Asp Ile Glu Asp Ser Leu Cys Gln Arg Lys Phe Asn Glu 290 295 300 Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys 305 310 315 320 Asp Arg Pro Ile Arg Ala Gln Pro Met Asn Pro Ser Val Thr Pro Leu 325 330 335 Ser Ser Leu Val Asp Ala Pro Thr Phe His Leu Gly Leu Arg Arg Thr 340 345 350 Glu Thr Glu Pro Thr Gly Leu Arg Leu Ser Asp Asn Lys Glu Val Pro 355 360 365 Val Cys Gly Asn Ser Thr Ser Lys Lys Arg Glu Arg Ser Phe Ser Gly 370 375 380 Pro Gly Val Leu Ser Arg Pro Ile Asn Thr Thr Pro Thr Met Asp Gln 385 390 395 400 Thr His Thr Arg Thr Trp Ser Gly Pro Cys Ile Tyr Ser Asn Val Cys 405 410 415 Thr Ile His Pro Asn Ser Ile Asn Glu Ser Thr Glu Gly His Ile Ser 420 425 430 Thr Ser Ala Glu Asp Lys Pro Val His Ser Arg Gly Trp Pro Arg Gly 435 440 445 Ile Lys Gly Trp Thr Arg Lys Ile Gly Asn Ala Met Arg Lys Leu Cys 450 455 460 Cys Cys Ile Pro Ser Lys Glu Thr Ser His Leu Gly Gln Arg Arg Val 465 470 475 480 Cys Pro Lys Ile 3 503 DNA Mus musculus 3 gtgtatcagt gtgtgtttgg gaggagcttg tgtgtgtgag ttgtgtttta agtttatttg 60 tgtgttagta cctttgggtt tgtgtgtgtg tctctgtgtg tttgtgtgtg tataactgtg 120 ggtgactgta agtgcacctg tgtgtttgta cgtgagtgtg taagactgtg tgtgtgcaca 180 agagcgtgtg taggtgctcg tgttgtaggt gtgagaacac ctgttgtgtt taggccatca 240 ttcagcttgg ccattgtttc taagctgcga gaccgggtca gatctaagat ggagagagac 300 atcctggcag aggtgaatca ccctttcatt gtcaagctgc attatgcctt tcagaccgaa 360 ggcaagctct acctgatcct ggacttcctg cggggaggtg acctcttcac caggctttcc 420 aaagaggtga tgttcacgga ggaggatgtc aagttctacc tggctgagct ggccttggct 480 ctagaccacc tccatggcct ggg 503 4 1590 DNA Mus musculus CDS (91)..(1542) 4 gtagcattta tactttgtta cctcaaatgg gctctgggag tcaacagaag tcagaaaagc 60 tcagatccaa gcccccttta tctgactgac atg gag aaa ttt cat gct caa tat 114 Met Glu Lys Phe His Ala Gln Tyr 1 5 gag atg cta gag act att ggc cag gga ggc tgc gca aag gtg aag ctg 162 Glu Met Leu Glu Thr Ile Gly Gln Gly Gly Cys Ala Lys Val Lys Leu 10 15 20 gcc cga cac cgc ctc aca ggc acc cac gtg gct gtc aaa atg att cca 210 Ala Arg His Arg Leu Thr Gly Thr His Val Ala Val Lys Met Ile Pro 25 30 35 40 aag agg gag tat tgg tgc aaa ctt ctg atg ttt gag gca gag tta ctg 258 Lys Arg Glu Tyr Trp Cys Lys Leu Leu Met Phe Glu Ala Glu Leu Leu 45 50 55 atg atg ttc aat cat cct aat atc atc tct ctc ctt caa gtc att gag 306 Met Met Phe Asn His Pro Asn Ile Ile Ser Leu Leu Gln Val Ile Glu 60 65 70 acc aag aag aaa gta tat ctc att atg gag ttg tgc gag ggt aaa tca 354 Thr Lys Lys Lys Val Tyr Leu Ile Met Glu Leu Cys Glu Gly Lys Ser 75 80 85 ctt tac caa cac atc caa aat gct ggc tac ctg cag gag gat gaa gca 402 Leu Tyr Gln His Ile Gln Asn Ala Gly Tyr Leu Gln Glu Asp Glu Ala 90 95 100 cgc cca tta ttc aag cag ctc tta agt gct atg aac tac tgc cac aac 450 Arg Pro Leu Phe Lys Gln Leu Leu Ser Ala Met Asn Tyr Cys His Asn 105 110 115 120 cag ggt ata gtt cac agg gac ctg aca cct gac aat att atg gta gaa 498 Gln Gly Ile Val His Arg Asp Leu Thr Pro Asp Asn Ile Met Val Glu 125 130 135 aaa gat ggg aga gtg aag aac att gat ttt gga ctc agc acc cac gtg 546 Lys Asp Gly Arg Val Lys Asn Ile Asp Phe Gly Leu Ser Thr His Val 140 145 150 aaa cca ggg caa aaa ctc aac tta ttc tgt ggg act tac cca ttt agt 594 Lys Pro Gly Gln Lys Leu Asn Leu Phe Cys Gly Thr Tyr Pro Phe Ser 155 160 165 gct cct gag gtg ctc ctt agc aga ccc tat ggt ggg ccc aag atc gat 642 Ala Pro Glu Val Leu Leu Ser Arg Pro Tyr Gly Gly Pro Lys Ile Asp 170 175 180 gta tgg act ctt gga gtt gtg ttg tat ttt atg gta att gga aag atc 690 Val Trp Thr Leu Gly Val Val Leu Tyr Phe Met Val Ile Gly Lys Ile 185 190 195 200 cca ttt gat gct gcc agc ata gaa aag ctg cgg aag caa att gtt gca 738 Pro Phe Asp Ala Ala Ser Ile Glu Lys Leu Arg Lys Gln Ile Val Ala 205 210 215 gga aag tat tct gct ccc tgt aga ctg tca gta aag ctt caa cac ctg 786 Gly Lys Tyr Ser Ala Pro Cys Arg Leu Ser Val Lys Leu Gln His Leu 220 225 230 att aat ctt tta atg acg gac aac ccc gaa ctt agg ccc act gtt gct 834 Ile Asn Leu Leu Met Thr Asp Asn Pro Glu Leu Arg Pro Thr Val Ala 235 240 245 gaa gtt atg gtg cat ccc tgg atc aca aaa ggc tca ggg gtg ttc cca 882 Glu Val Met Val His Pro Trp Ile Thr Lys Gly Ser Gly Val Phe Pro 250 255 260 gat cct tgt gaa gaa cag ata ccc ctc aag cca gac cct gcg att gta 930 Asp Pro Cys Glu Glu Gln Ile Pro Leu Lys Pro Asp Pro Ala Ile Val 265 270 275 280 aaa cca atg gga cat att ggg ttc caa gct caa gac att gaa gat tcg 978 Lys Pro Met Gly His Ile Gly Phe Gln Ala Gln Asp Ile Glu Asp Ser 285 290 295 tta cgt cag aga aaa ttc aat gaa acc atg gca tct tat tgt cta ctg 1026 Leu Arg Gln Arg Lys Phe Asn Glu Thr Met Ala Ser Tyr Cys Leu Leu 300 305 310 aaa aaa cag att ctt aag gaa tgt gac agg cca atc cgg gat cag ccc 1074 Lys Lys Gln Ile Leu Lys Glu Cys Asp Arg Pro Ile Arg Asp Gln Pro 315 320 325 atg aat cca tca gtg acc cca ttc cct tcc ctt gtt gat act cct act 1122 Met Asn Pro Ser Val Thr Pro Phe Pro Ser Leu Val Asp Thr Pro Thr 330 335 340 ttc cat ctc gga ctt cgg agg aga gag act gaa ccc aca ggt ctc aga 1170 Phe His Leu Gly Leu Arg Arg Arg Glu Thr Glu Pro Thr Gly Leu Arg 345 350 355 360 tta tct gcc aat agg caa gtg tct gtc tgt gga aaa agt aca agt aag 1218 Leu Ser Ala Asn Arg Gln Val Ser Val Cys Gly Lys Ser Thr Ser Lys 365 370 375 aaa aga gac aga agt ttc att tgg ccc ggt gtt ctc agc agg ccg att 1266 Lys Arg Asp Arg Ser Phe Ile Trp Pro Gly Val Leu Ser Arg Pro Ile 380 385 390 aac aca aca ccc aca atg gac caa aca cac acc cgt act agg agt gtt 1314 Asn Thr Thr Pro Thr Met Asp Gln Thr His Thr Arg Thr Arg Ser Val 395 400 405 ccc tgc att tac tca aat gtt tgc aca atc cat cca aac agc atc gat 1362 Pro Cys Ile Tyr Ser Asn Val Cys Thr Ile His Pro Asn Ser Ile Asp 410 415 420 gag agt aca gaa ggc cac acc agt gcc tca gca gag gat aag cct gtc 1410 Glu Ser Thr Glu Gly His Thr Ser Ala Ser Ala Glu Asp Lys Pro Val 425 430 435 440 cac agc aga ggc tgg ccc aga ggc atc aag ggc tgg act agg aag ata 1458 His Ser Arg Gly Trp Pro Arg Gly Ile Lys Gly Trp Thr Arg Lys Ile 445 450 455 gga aat gca atg agg aag ctc tgt tgc tgt atc cca tcc aaa gag aca 1506 Gly Asn Ala Met Arg Lys Leu Cys Cys Cys Ile Pro Ser Lys Glu Thr 460 465 470 tct cac ctg ggg cag agc aga gtc tgc cca aaa aaa taagacacag 1552 Ser His Leu Gly Gln Ser Arg Val Cys Pro Lys Lys 475 480 gaagggtgtc aggagaacga gcatccggca cggcccag 1590 5 484 PRT Mus musculus 5 Met Glu Lys Phe His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln 1 5 10 15 Gly Gly Cys Ala Lys Val Lys Leu Ala Arg His Arg Leu Thr Gly Thr 20 25 30 His Val Ala Val Lys Met Ile Pro Lys Arg Glu Tyr Trp Cys Lys Leu 35 40 45 Leu Met Phe Glu Ala Glu Leu Leu Met Met Phe Asn His Pro Asn Ile 50 55 60 Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile 65 70 75 80 Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala 85 90 95 Gly Tyr Leu Gln Glu Asp Glu Ala Arg Pro Leu Phe Lys Gln Leu Leu 100 105 110 Ser Ala Met Asn Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu 115 120 125 Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Arg Val Lys Asn Ile 130 135 140 Asp Phe Gly Leu Ser Thr His Val Lys Pro Gly Gln Lys Leu Asn Leu 145 150 155 160 Phe Cys Gly Thr Tyr Pro Phe Ser Ala Pro Glu Val Leu Leu Ser Arg 165 170 175 Pro Tyr Gly Gly Pro Lys Ile Asp Val Trp Thr Leu Gly Val Val Leu 180 185 190 Tyr Phe Met Val Ile Gly Lys Ile Pro Phe Asp Ala Ala Ser Ile Glu 195 200 205 Lys Leu Arg Lys Gln Ile Val Ala Gly Lys Tyr Ser Ala Pro Cys Arg 210 215 220 Leu Ser Val Lys Leu Gln His Leu Ile Asn Leu Leu Met Thr Asp Asn 225 230 235 240 Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val His Pro Trp Ile 245 250 255 Thr Lys Gly Ser Gly Val Phe Pro Asp Pro Cys Glu Glu Gln Ile Pro 260 265 270 Leu Lys Pro Asp Pro Ala Ile Val Lys Pro Met Gly His Ile Gly Phe 275 280 285 Gln Ala Gln Asp Ile Glu Asp Ser Leu Arg Gln Arg Lys Phe Asn Glu 290 295 300 Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys 305 310 315 320 Asp Arg Pro Ile Arg Asp Gln Pro Met Asn Pro Ser Val Thr Pro Phe 325 330 335 Pro Ser Leu Val Asp Thr Pro Thr Phe His Leu Gly Leu Arg Arg Arg 340 345 350 Glu Thr Glu Pro Thr Gly Leu Arg Leu Ser Ala Asn Arg Gln Val Ser 355 360 365 Val Cys Gly Lys Ser Thr Ser Lys Lys Arg Asp Arg Ser Phe Ile Trp 370 375 380 Pro Gly Val Leu Ser Arg Pro Ile Asn Thr Thr Pro Thr Met Asp Gln 385 390 395 400 Thr His Thr Arg Thr Arg Ser Val Pro Cys Ile Tyr Ser Asn Val Cys 405 410 415 Thr Ile His Pro Asn Ser Ile Asp Glu Ser Thr Glu Gly His Thr Ser 420 425 430 Ala Ser Ala Glu Asp Lys Pro Val His Ser Arg Gly Trp Pro Arg Gly 435 440 445 Ile Lys Gly Trp Thr Arg Lys Ile Gly Asn Ala Met Arg Lys Leu Cys 450 455 460 Cys Cys Ile Pro Ser Lys Glu Thr Ser His Leu Gly Gln Ser Arg Val 465 470 475 480 Cys Pro Lys Lys 6 2257 DNA Mus musculus CDS (434)..(1798) 6 ggagtttggt ggagttggtg gagttggtgg tgccctttgt gatttcgttg tatctagtga 60 gttgtgtgtg tgtgtgtgtg tgtgtgtgtg tagttcagtg tgtgtttggg aggagcttgt 120 gtgtgtgagt tgtgaattaa gtttacttgc gtgtgaatac ctttgtgttt ttgtgtgtgt 180 ctgtgtgtat ccatgtgggt gactgtaagt gcacctgtgt gatagttcga aagtgtatga 240 gagagtgtgt gtgggcacaa gagtgtgtgt aggtgcacgt gtggtaggtg tgagaacacc 300 tcttgtgttg aggccgtcag tcagcttggc cattgtttct aaggtagcat ttatactttg 360 ttacctcaaa tgggctctgg gagtcaacag aagtcagaaa agctcagatc caagccccct 420 ttatctgact gac atg gag aaa ttt cat gct caa tat gag atg cta gag 469 Met Glu Lys Phe His Ala Gln Tyr Glu Met Leu Glu 1 5 10 act att ggc cag gga ggc tgc gca aag gtg aag ctg gcc cga cac cgc 517 Thr Ile Gly Gln Gly Gly Cys Ala Lys Val Lys Leu Ala Arg His Arg 15 20 25 ctc aca ggc acc cac gtg gct gtc aaa atg att cca aag agg gag tat 565 Leu Thr Gly Thr His Val Ala Val Lys Met Ile Pro Lys Arg Glu Tyr 30 35 40 tgg tgc aaa ctt ctg atg ttt gag gca gag tta ctg atg atg ttc aat 613 Trp Cys Lys Leu Leu Met Phe Glu Ala Glu Leu Leu Met Met Phe Asn 45 50 55 60 cat cct aat atc atc tct ctc ctt caa gtc att gag acc aag aag aaa 661 His Pro Asn Ile Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys 65 70 75 gta tat ctc att atg gag ttg tgc gag ggt aaa tca ctt tac caa cac 709 Val Tyr Leu Ile Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His 80 85 90 atc caa aat gct ggc tac ctg cag gag gat gaa gca cgc cca tta ttc 757 Ile Gln Asn Ala Gly Tyr Leu Gln Glu Asp Glu Ala Arg Pro Leu Phe 95 100 105 aag cag ctc tta agt gct atg aac tac tgc cac aac cag ggt ata gtt 805 Lys Gln Leu Leu Ser Ala Met Asn Tyr Cys His Asn Gln Gly Ile Val 110 115 120 cac agg gac ctg aca cct gac aat att atg gta gaa aaa gat ggg aga 853 His Arg Asp Leu Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Arg 125 130 135 140 gtg aag aac att gat ttt gga ctc agc acc cac gtg aaa cca ggg caa 901 Val Lys Asn Ile Asp Phe Gly Leu Ser Thr His Val Lys Pro Gly Gln 145 150 155 aaa ctc aac tta ttc tgt ggg act tac cca ttt agt gct cct gag gtg 949 Lys Leu Asn Leu Phe Cys Gly Thr Tyr Pro Phe Ser Ala Pro Glu Val 160 165 170 ctc ctt agc aga ccc tat ggt ggg ccc aag atc gat gta tgg act ctt 997 Leu Leu Ser Arg Pro Tyr Gly Gly Pro Lys Ile Asp Val Trp Thr Leu 175 180 185 gga gtt gtg ttg tat ttt atg gta att gga aag atc cca ttt gat gct 1045 Gly Val Val Leu Tyr Phe Met Val Ile Gly Lys Ile Pro Phe Asp Ala 190 195 200 gcc agc ata gaa aag ctg cgg aag caa att gtt gca gga aag tat tct 1093 Ala Ser Ile Glu Lys Leu Arg Lys Gln Ile Val Ala Gly Lys Tyr Ser 205 210 215 220 gct ccc tgt aga ctg tca gta aag ctt caa cac ctg att aat ctt tta 1141 Ala Pro Cys Arg Leu Ser Val Lys Leu Gln His Leu Ile Asn Leu Leu 225 230 235 atg acg gac aac ccc gaa ctt agg ccc act gtt gct gaa gtt atg gtg 1189 Met Thr Asp Asn Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val 240 245 250 cat ccc tgg atc aca aaa ggc tca ggg gtg ttc cca gat cct tgt gaa 1237 His Pro Trp Ile Thr Lys Gly Ser Gly Val Phe Pro Asp Pro Cys Glu 255 260 265 gaa cag ata ccc ctc aag cca gac cct gcg att gta aaa cca atg gga 1285 Glu Gln Ile Pro Leu Lys Pro Asp Pro Ala Ile Val Lys Pro Met Gly 270 275 280 cat att ggg ttc caa gct caa gac att gaa gat tcg tta cgt cag aga 1333 His Ile Gly Phe Gln Ala Gln Asp Ile Glu Asp Ser Leu Arg Gln Arg 285 290 295 300 aaa ttc aat gaa acc atg gca tct tat tgt cta ctg aaa aaa cag att 1381 Lys Phe Asn Glu Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile 305 310 315 ctt aag gaa tgt gac agg cca atc cgg gat cag ccc atg aat cca tca 1429 Leu Lys Glu Cys Asp Arg Pro Ile Arg Asp Gln Pro Met Asn Pro Ser 320 325 330 gtg acc cca ttc cct tcc ctt gtt gat act cct act ttc cat ctc gga 1477 Val Thr Pro Phe Pro Ser Leu Val Asp Thr Pro Thr Phe His Leu Gly 335 340 345 ctt cgg agg aga gag act gaa ccc aca ggc tca gat tat ctg cca ata 1525 Leu Arg Arg Arg Glu Thr Glu Pro Thr Gly Ser Asp Tyr Leu Pro Ile 350 355 360 ggc aag tgt ctg tct gtg gaa aaa gta caa gta aga aaa gag aca gaa 1573 Gly Lys Cys Leu Ser Val Glu Lys Val Gln Val Arg Lys Glu Thr Glu 365 370 375 380 gtt tca ttt ggc ccg gtg ttc tca gca ggc cga tta aca caa cac cca 1621 Val Ser Phe Gly Pro Val Phe Ser Ala Gly Arg Leu Thr Gln His Pro 385 390 395 caa tgg acc aaa cac aca ccc gta cta gga gtg ttc cct gca ttt act 1669 Gln Trp Thr Lys His Thr Pro Val Leu Gly Val Phe Pro Ala Phe Thr 400 405 410 caa atg ttt gca caa tcc atc caa aca gca tcg atg aga gta cag aag 1717 Gln Met Phe Ala Gln Ser Ile Gln Thr Ala Ser Met Arg Val Gln Lys 415 420 425 gcc aca cca gtg cct cag cag agg ata agc ctg tcc aca gca gag gct 1765 Ala Thr Pro Val Pro Gln Gln Arg Ile Ser Leu Ser Thr Ala Glu Ala 430 435 440 ggc cca gag gca tca agg gct gga cta gga aga taggaaatgc aatgaggaag 1818 Gly Pro Glu Ala Ser Arg Ala Gly Leu Gly Arg 445 450 455 ctctgttgct gtatcccatc caaagagaca tctcacctgg ggcagagcag agtctgccca 1878 aaaaaataag acacaggaag ggtgtcagga gaacgagcat ccggcacggc ccagaagatc 1938 accagaggat gccggatgct acgattcaac agttataata ttggaaagga cccatgtata 1998 gacatggacc tgcaaaaggg aaccttgtgg aaaggcatca tgttctgggt tcagcatgtt 2058 tcactcagag ccccgggtcc agccaggggg aagaaagcaa atgatgaaat cccagatggt 2118 gtctgggatc accattcaga gcaggggctg aaagcctgtc caaagctggt agagacagaa 2178 gcccctctgc ctacccaggg tcataatcag actcctgctc tgagaataaa atagatgttt 2238 gtgaaagatg aaaaaaaaa 2257 7 455 PRT Mus musculus 7 Met Glu Lys Phe His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln 1 5 10 15 Gly Gly Cys Ala Lys Val Lys Leu Ala Arg His Arg Leu Thr Gly Thr 20 25 30 His Val Ala Val Lys Met Ile Pro Lys Arg Glu Tyr Trp Cys Lys Leu 35 40 45 Leu Met Phe Glu Ala Glu Leu Leu Met Met Phe Asn His Pro Asn Ile 50 55 60 Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile 65 70 75 80 Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala 85 90 95 Gly Tyr Leu Gln Glu Asp Glu Ala Arg Pro Leu Phe Lys Gln Leu Leu 100 105 110 Ser Ala Met Asn Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu 115 120 125 Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Arg Val Lys Asn Ile 130 135 140 Asp Phe Gly Leu Ser Thr His Val Lys Pro Gly Gln Lys Leu Asn Leu 145 150 155 160 Phe Cys Gly Thr Tyr Pro Phe Ser Ala Pro Glu Val Leu Leu Ser Arg 165 170 175 Pro Tyr Gly Gly Pro Lys Ile Asp Val Trp Thr Leu Gly Val Val Leu 180 185 190 Tyr Phe Met Val Ile Gly Lys Ile Pro Phe Asp Ala Ala Ser Ile Glu 195 200 205 Lys Leu Arg Lys Gln Ile Val Ala Gly Lys Tyr Ser Ala Pro Cys Arg 210 215 220 Leu Ser Val Lys Leu Gln His Leu Ile Asn Leu Leu Met Thr Asp Asn 225 230 235 240 Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val His Pro Trp Ile 245 250 255 Thr Lys Gly Ser Gly Val Phe Pro Asp Pro Cys Glu Glu Gln Ile Pro 260 265 270 Leu Lys Pro Asp Pro Ala Ile Val Lys Pro Met Gly His Ile Gly Phe 275 280 285 Gln Ala Gln Asp Ile Glu Asp Ser Leu Arg Gln Arg Lys Phe Asn Glu 290 295 300 Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys 305 310 315 320 Asp Arg Pro Ile Arg Asp Gln Pro Met Asn Pro Ser Val Thr Pro Phe 325 330 335 Pro Ser Leu Val Asp Thr Pro Thr Phe His Leu Gly Leu Arg Arg Arg 340 345 350 Glu Thr Glu Pro Thr Gly Ser Asp Tyr Leu Pro Ile Gly Lys Cys Leu 355 360 365 Ser Val Glu Lys Val Gln Val Arg Lys Glu Thr Glu Val Ser Phe Gly 370 375 380 Pro Val Phe Ser Ala Gly Arg Leu Thr Gln His Pro Gln Trp Thr Lys 385 390 395 400 His Thr Pro Val Leu Gly Val Phe Pro Ala Phe Thr Gln Met Phe Ala 405 410 415 Gln Ser Ile Gln Thr Ala Ser Met Arg Val Gln Lys Ala Thr Pro Val 420 425 430 Pro Gln Gln Arg Ile Ser Leu Ser Thr Ala Glu Ala Gly Pro Glu Ala 435 440 445 Ser Arg Ala Gly Leu Gly Arg 450 455 8 2596 DNA Mus musculus 8 gaattcccgg gtcgacccac gcgtccggca ggaattcaca gagttaccgt gcttgcctct 60 gtggaagtgg gtcaagctga tggtatctaa tattctctct ggtccttctg atcatgctgc 120 tgggtccaga agtacccaga ttccacaccc agcttctaca ctcccccact tcaggtacct 180 gaaagcttgg tcccttcaaa ggcactttta atgatctggt ggtttggggt gtgaagttat 240 tctacctggg gcttttgtac accacaggaa caattttcct cttacttctc ccacttcctc 300 tccctagcat ggtcagttct cctccttgtt caacgtgcat gatacacaca ggagatactt 360 tctgggatgt tagatctgtt ggcaggttcg atttaaccac catcccatgg tgtctagacc 420 tagcttcccc atgcatcaca ccatatacat atacataagt ataatctgcc agtttacaca 480 gacatgagta acatagatac attcaaatac agaaatgtac ctgggccgtg ccagatgctc 540 attctcctga caaccttcct gtgtcttatt ttttggggag actctgctgt gccccacgtg 600 agctgtctct tttgatggga tacagcaaca gagtttcctc attgcatttc ctatcttcct 660 agtccagccc ttgatgcctc tgggccagcc tctgctgcgg acaggcttat cctctgctga 720 ggcactggtg tggccttctg tactctcatc actgctattt ggatggattg tgcaaacatt 780 tgagtaaatg cagggaacac tcctagtatg ggtgtgtctg tggtccattg tggatgttat 840 gtggagtggc ctgccggaaa cactgggcca actgaatctt ctgtctcttt ttttactagt 900 acacttgcca cagacagaca cttgcctatt gccagatgac cagagacctg tgggttcagt 960 ctctcccctc caaagtctga gatggaaagt aggagtatca acaagggaag ggaatggggt 1020 cagcgacaga ttcatgggct gagcccggat tggcctgtca cattccttaa gaatctgttt 1080 tttcagtaga caataagatg ccatggtttc attgaatttt ctctgacata acgaatcttc 1140 aatgtcttga gcttggaacc cgatatatcc cattgctttt acaatcgcag ggtctggctt 1200 gaggggtatc tgttcttcac aaggatctgg gaacacgcct gagccttctg tgacccaggg 1260 atgcaccata acttcagcaa cagtgggcct aagttcgggg ttgtccgtca ttaaaagtct 1320 aatcaggtct tggagctcta ctgacagtct acagggaaca gaacactttc ctgcaacaat 1380 ttgctttcgc agcttttcta tgctggcagc atcaaacaga atctttccag ttaccataaa 1440 atacagcaca actctaagac cccacacatc gatcttgcgc atatcatagg gtctgctaag 1500 gagcacctca ggagtactaa atgggtaagt ctcacagaat aagttgagtt tttgccctgg 1560 cttctcttgg gtgccgagtc caaaatcaat gatcttcact ttcccatctt tttctaccat 1620 aatattgtca ggtgtcaggt ccctggttgt ggcagtagtt catagcactt aagagctgct 1680 tgaataatgg gcgtgcttca tcctcctgca ggtagccagc gtttcagatg tgttggtaaa 1740 gtgatttacc ctctcacaac tccataatga ggtatacttt cttcttggtc tcaatgactt 1800 gaaggagaga gatgatattt ggatgatcgg ccatcatcag taactctgcc tcagacatga 1860 cagggttgca ccaatactcc ctctttcgaa tcactttgac agccacgtgg gtgcctgtga 1920 ggcggtgcct ggccagcttc accttggcac agcctccatc gccgatagtc tctagcatct 1980 catattgagc atgaaatttc gccatgtcag agaaaggggg cttggatctg agcttttctg 2040 atttctgttg actcccagag cccacttgag gtaacaaagt ataaatgcta cctaaggggg 2100 cggggagaaa taaagggaag aaagaaaggt aagataaaaa ttaaaatagt gaaaaataag 2160 caaaacagaa aattaaaacc caacaaaaaa taataacagc agaaacccag aagagcaaaa 2220 ccacacacaa agccaagaaa atccaaatta aaaaacctag ctgcaagtcc ctaggagaga 2280 ggggcacagc tcagcaacac taagaagaaa tattactaag tgaggagcca agtgtgttgg 2340 cgcacacctt taatcccctg actcgggagg ccgaggcagg tggatttctg agttcggggc 2400 cagcctggtc tacagagtga attccaggac agccagagct atacagagaa atcctatctc 2460 aaaaaacaaa caaacaaaca aacaaaaaac tctactagga aatatataaa tgattagtat 2520 aacaaactca tcaaaacttc tagaatatac aaagaactaa aaaaaaaaaa aaaagggcgg 2580 ccgctctaga ggatcc 2596 9 2738 DNA Mus musculus CDS (439)..(1950) 9 agttggtgga gttggtggag tttggtggag ttggtggtgc cctttgcgat ttcgttgtat 60 ctagtgagct gtgtgtggat tttgtgtttg attggtttgt gtgtgagctt gtgtgtgtgt 120 gtgtgtgtgt gtgtgtgtgt gtgtctagat cagtgtgtgt ttgggaggag cgttgcttgt 180 gtttgtgagt tgagttttaa gtttacttgc gtgtgagtaa ctttgtgttg tgtgtgggtg 240 tgtgtgtgta ggtatacccg tgggtgactc taagtgcacc tgtgtgtttg tgaccgagtg 300 tgtgagagtg tgtgtgtgtg agcacacaag agtgtgtgta ggtgcacgtg tagcaggtgt 360 gagaacatct gttgtgttga ggccgtcagt cagcttggcc attgtttcta aggtagcatt 420 tatacttggt tacctcaa atg ggc cct ggg agt caa cag aag tca gaa aag 471 Met Gly Pro Gly Ser Gln Gln Lys Ser Glu Lys 1 5 10 ctc aga tcc aag tcc cct ttg gct gac atg gat ggt ttg cat gct caa 519 Leu Arg Ser Lys Ser Pro Leu Ala Asp Met Asp Gly Leu His Ala Gln 15 20 25 tat gtg atg cta gag act atc ggc cat gga ggc tgt gcc aca gtg aag 567 Tyr Val Met Leu Glu Thr Ile Gly His Gly Gly Cys Ala Thr Val Lys 30 35 40 ctg gcc cag cac cgc ctc aca ggc act cac gtg gct gtc aaa acg att 615 Leu Ala Gln His Arg Leu Thr Gly Thr His Val Ala Val Lys Thr Ile 45 50 55 cga aag agg gag tat tgg tgc aac cgt gtc att tct gag gta gag tta 663 Arg Lys Arg Glu Tyr Trp Cys Asn Arg Val Ile Ser Glu Val Glu Leu 60 65 70 75 ctg atg atg gcc gat cat ccg aat atc atc tct ctc ctt caa gtc att 711 Leu Met Met Ala Asp His Pro Asn Ile Ile Ser Leu Leu Gln Val Ile 80 85 90 gag acc aag aag aaa gta tac ctc att atg gag ttg tgc aag ggt aaa 759 Glu Thr Lys Lys Lys Val Tyr Leu Ile Met Glu Leu Cys Lys Gly Lys 95 100 105 tca ctt tac caa cac atc cga aaa gct ggc tac ctg cag gag cat gaa 807 Ser Leu Tyr Gln His Ile Arg Lys Ala Gly Tyr Leu Gln Glu His Glu 110 115 120 gca cgc gca tta ttc aag cag ctc tta agt gct atg aac tac tgc cac 855 Ala Arg Ala Leu Phe Lys Gln Leu Leu Ser Ala Met Asn Tyr Cys His 125 130 135 aac cag ggt ata gtt cac agg gac ctg aaa ccg gac aat atc atg gtt 903 Asn Gln Gly Ile Val His Arg Asp Leu Lys Pro Asp Asn Ile Met Val 140 145 150 155 gaa aaa gat ggg aaa gtg aag atc att gat ttt gga ctc ggc acc aaa 951 Glu Lys Asp Gly Lys Val Lys Ile Ile Asp Phe Gly Leu Gly Thr Lys 160 165 170 gtg aag cca ggg caa aaa ctc aac tta ttc tgt ggg act tac cca ttt 999 Val Lys Pro Gly Gln Lys Leu Asn Leu Phe Cys Gly Thr Tyr Pro Phe 175 180 185 agt gct cct gag gtg ctc ctt agc aca ccc tat gat ggg ccc aag atc 1047 Ser Ala Pro Glu Val Leu Leu Ser Thr Pro Tyr Asp Gly Pro Lys Ile 190 195 200 gat gta tgg act ctt gga gtt gtg ctg tat ttt atg gta act gga aag 1095 Asp Val Trp Thr Leu Gly Val Val Leu Tyr Phe Met Val Thr Gly Lys 205 210 215 atc ccg ttt gat gct tgc agc ata aaa aag ctg gta aag cga att ctt 1143 Ile Pro Phe Asp Ala Cys Ser Ile Lys Lys Leu Val Lys Arg Ile Leu 220 225 230 235 gca gga aag tat tct att ccc tct aga ctg tca gca gag ctc caa gac 1191 Ala Gly Lys Tyr Ser Ile Pro Ser Arg Leu Ser Ala Glu Leu Gln Asp 240 245 250 ctg ctt agt ctt tta atg acg gcc aac ccc aaa ctc agg ccc act gtt 1239 Leu Leu Ser Leu Leu Met Thr Ala Asn Pro Lys Leu Arg Pro Thr Val 255 260 265 gct gag gtt atg gtg cat ccc tgg gtc aca gaa ggc tca ggg gtg ttc 1287 Ala Glu Val Met Val His Pro Trp Val Thr Glu Gly Ser Gly Val Phe 270 275 280 cca gat cct tgt gaa gaa cag acc ccc ctc aag cca gac cct gca att 1335 Pro Asp Pro Cys Glu Glu Gln Thr Pro Leu Lys Pro Asp Pro Ala Ile 285 290 295 gta aaa gca atg gga cat atc ggg ttc caa gct caa gat att gaa gat 1383 Val Lys Ala Met Gly His Ile Gly Phe Gln Ala Gln Asp Ile Glu Asp 300 305 310 315 tcg tta cgt cag aga aaa ttc aac caa acc atg gcg tct tat tgt cta 1431 Ser Leu Arg Gln Arg Lys Phe Asn Gln Thr Met Ala Ser Tyr Cys Leu 320 325 330 ctg aaa aaa cag att ctt aag gaa tgt gac agg cca acc cgg gct agg 1479 Leu Lys Lys Gln Ile Leu Lys Glu Cys Asp Arg Pro Thr Arg Ala Arg 335 340 345 ccc gtg aac cca tcg gtg acc cca ttc cct tcc ctt gtt gat act gct 1527 Pro Val Asn Pro Ser Val Thr Pro Phe Pro Ser Leu Val Asp Thr Ala 350 355 360 act acc cgt ctc gga ctt cgc agg aga gag aat gaa ccc aca tgt ccc 1575 Thr Thr Arg Leu Gly Leu Arg Arg Arg Glu Asn Glu Pro Thr Cys Pro 365 370 375 tgg tca tcc gcc aat agg caa gtg tct gtc tgt ggc aag agt act agt 1623 Trp Ser Ser Ala Asn Arg Gln Val Ser Val Cys Gly Lys Ser Thr Ser 380 385 390 395 aag aaa aga gac aga aga gtc agt tgg ccc agt gtt ctc ggc agg cca 1671 Lys Lys Arg Asp Arg Arg Val Ser Trp Pro Ser Val Leu Gly Arg Pro 400 405 410 cgc cac acg gca ccc aca atg gac cac aca cgc acc cgt act agg agt 1719 Arg His Thr Ala Pro Thr Met Asp His Thr Arg Thr Arg Thr Arg Ser 415 420 425 gta ccc tgc att tgc tca atg ttt tgc aca gtc cag cca aac agc agc 1767 Val Pro Cys Ile Cys Ser Met Phe Cys Thr Val Gln Pro Asn Ser Ser 430 435 440 gaa gag agc aca caa ggc cac acc aga gcc tca gca gca gat aag cct 1815 Glu Glu Ser Thr Gln Gly His Thr Arg Ala Ser Ala Ala Asp Lys Pro 445 450 455 gtc cac agc agg ggc tgg ccc aga ggc atc aag ggc tgg acg agg atg 1863 Val His Ser Arg Gly Trp Pro Arg Gly Ile Lys Gly Trp Thr Arg Met 460 465 470 475 ata gga aat gcg atg agg aag ctc tgt tgc tgt atc cca tcc aaa gag 1911 Ile Gly Asn Ala Met Arg Lys Leu Cys Cys Cys Ile Pro Ser Lys Glu 480 485 490 aca tct cac ctg ggg cag aac aga gtc tcc ccc aaa aaa taagacacag 1960 Thr Ser His Leu Gly Gln Asn Arg Val Ser Pro Lys Lys 495 500 gaagggtgtc aggagaacaa gcatccggca cggcccaggt acatttctgc atttgaatgt 2020 atctatgtta ctcatgtctg tgtcaactgg cagatgatac ttatgtatat ggtgcaaagc 2080 atggggaagc taggtgtaga caccgtggga tgatgggtaa atcgaacctg ccaacagacc 2140 tagcatccca gaaggtatct cctgcgtgta tcctgcatgt tgaacaagga ggggaactga 2200 ccatgctagg gggaggaagt gggagaagga agaggaggag atgctgaggg aggagaggat 2260 ggtatgtgat gggagctagg agatgggggg aagaggttga gacaggagga ggcaacttgg 2320 gggagcagtg tgaaacaggg taaccacagc tggagagatg ccctgtgcag ctgaggttct 2380 cagagtccct ctcacgtgtg ctttgcattt tagaagatca ccagaggatg ccggatgcta 2440 cgattctaca gttatagtat tggaaaggac ccgtgtatag acacggacct gaaaaaggga 2500 accttgtgga aaggcatcat gttctgggtt cagcgtgctt cactcagagc ccccagtcca 2560 gccagggggc aagaaagcaa atgatgaaat cccagatggg ctctgggatc accattcaga 2620 gaagtggctt aaagcatgtc caaagctgat agagacagcc cctctgcctg cccaagctca 2680 taatcagact cctcctctga gaataaaata gatgtttgtg aaaaaaaaaa aaaaaaaa 2738 10 504 PRT Mus musculus 10 Met Gly Pro Gly Ser Gln Gln Lys Ser Glu Lys Leu Arg Ser Lys Ser 1 5 10 15 Pro Leu Ala Asp Met Asp Gly Leu His Ala Gln Tyr Val Met Leu Glu 20 25 30 Thr Ile Gly His Gly Gly Cys Ala Thr Val Lys Leu Ala Gln His Arg 35 40 45 Leu Thr Gly Thr His Val Ala Val Lys Thr Ile Arg Lys Arg Glu Tyr 50 55 60 Trp Cys Asn Arg Val Ile Ser Glu Val Glu Leu Leu Met Met Ala Asp 65 70 75 80 His Pro Asn Ile Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys 85 90 95 Val Tyr Leu Ile Met Glu Leu Cys Lys Gly Lys Ser Leu Tyr Gln His 100 105 110 Ile Arg Lys Ala Gly Tyr Leu Gln Glu His Glu Ala Arg Ala Leu Phe 115 120 125 Lys Gln Leu Leu Ser Ala Met Asn Tyr Cys His Asn Gln Gly Ile Val 130 135 140 His Arg Asp Leu Lys Pro Asp Asn Ile Met Val Glu Lys Asp Gly Lys 145 150 155 160 Val Lys Ile Ile Asp Phe Gly Leu Gly Thr Lys Val Lys Pro Gly Gln 165 170 175 Lys Leu Asn Leu Phe Cys Gly Thr Tyr Pro Phe Ser Ala Pro Glu Val 180 185 190 Leu Leu Ser Thr Pro Tyr Asp Gly Pro Lys Ile Asp Val Trp Thr Leu 195 200 205 Gly Val Val Leu Tyr Phe Met Val Thr Gly Lys Ile Pro Phe Asp Ala 210 215 220 Cys Ser Ile Lys Lys Leu Val Lys Arg Ile Leu Ala Gly Lys Tyr Ser 225 230 235 240 Ile Pro Ser Arg Leu Ser Ala Glu Leu Gln Asp Leu Leu Ser Leu Leu 245 250 255 Met Thr Ala Asn Pro Lys Leu Arg Pro Thr Val Ala Glu Val Met Val 260 265 270 His Pro Trp Val Thr Glu Gly Ser Gly Val Phe Pro Asp Pro Cys Glu 275 280 285 Glu Gln Thr Pro Leu Lys Pro Asp Pro Ala Ile Val Lys Ala Met Gly 290 295 300 His Ile Gly Phe Gln Ala Gln Asp Ile Glu Asp Ser Leu Arg Gln Arg 305 310 315 320 Lys Phe Asn Gln Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile 325 330 335 Leu Lys Glu Cys Asp Arg Pro Thr Arg Ala Arg Pro Val Asn Pro Ser 340 345 350 Val Thr Pro Phe Pro Ser Leu Val Asp Thr Ala Thr Thr Arg Leu Gly 355 360 365 Leu Arg Arg Arg Glu Asn Glu Pro Thr Cys Pro Trp Ser Ser Ala Asn 370 375 380 Arg Gln Val Ser Val Cys Gly Lys Ser Thr Ser Lys Lys Arg Asp Arg 385 390 395 400 Arg Val Ser Trp Pro Ser Val Leu Gly Arg Pro Arg His Thr Ala Pro 405 410 415 Thr Met Asp His Thr Arg Thr Arg Thr Arg Ser Val Pro Cys Ile Cys 420 425 430 Ser Met Phe Cys Thr Val Gln Pro Asn Ser Ser Glu Glu Ser Thr Gln 435 440 445 Gly His Thr Arg Ala Ser Ala Ala Asp Lys Pro Val His Ser Arg Gly 450 455 460 Trp Pro Arg Gly Ile Lys Gly Trp Thr Arg Met Ile Gly Asn Ala Met 465 470 475 480 Arg Lys Leu Cys Cys Cys Ile Pro Ser Lys Glu Thr Ser His Leu Gly 485 490 495 Gln Asn Arg Val Ser Pro Lys Lys 500 11 2827 DNA Mus musculus CDS (524)..(1975) 11 gagaggagtt ggtggagttg gtggagtttg gtggatttgg tggagttggt ggtgcccttt 60 gcgatttcgt tgtatctagt gagccgtgtg tggattttgt gtttgattgg ttcgtgtgtg 120 agcttttgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtagatc 180 agtgtgtgtt tgggaggagc ttgtgtgtgt gagttgtgtt ttaagtttat ttgcgtgtga 240 gtacctttgg gtttttgtgt gtgtctgtgt gtgtttgtgt gtgtataact gtgggtgact 300 gtaagtgcac ctgtgtgttt gtacgtgagt gtgtaagact gtgtgtgtgc acaagagcgt 360 gtgtaggtgc acgtgttgta ggtgtgagaa cacctgttgt gtttaggcca tcagtcagct 420 tggtcattgt ttctaaggta gcatttatac tttgttacct caagtgggct ctgggagtca 480 acagaagtca gaaaagctca gatccaagcc ccctttttct gac atg gag aaa ttt 535 Met Glu Lys Phe 1 cat gct caa tat gag atg cta gag act att ggc cag gga ggc tgc gcc 583 His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln Gly Gly Cys Ala 5 10 15 20 cag gtg aag ctg gcc cga cac cgc ctc aca ggc acc cac gtg gct gtc 631 Gln Val Lys Leu Ala Arg His Arg Leu Thr Gly Thr His Val Ala Val 25 30 35 aaa gtg att gta aag agg gag tgt tgg ttc aac cct gtc atg tct gag 679 Lys Val Ile Val Lys Arg Glu Cys Trp Phe Asn Pro Val Met Ser Glu 40 45 50 gca gag tta ctg atg atg acc gat cat ccg aat atc atc tct ctc ctt 727 Ala Glu Leu Leu Met Met Thr Asp His Pro Asn Ile Ile Ser Leu Leu 55 60 65 caa gtc att gag acc aag aag aaa gta tac ctc att atg gag ttg tgc 775 Gln Val Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile Met Glu Leu Cys 70 75 80 gag ggt aaa tca ctt tac caa cac atc caa aat gct ggc tac ctg cag 823 Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala Gly Tyr Leu Gln 85 90 95 100 gag gat gaa gca cgc cca tta ttc aag cag ctc tta agt gct atg aac 871 Glu Asp Glu Ala Arg Pro Leu Phe Lys Gln Leu Leu Ser Ala Met Asn 105 110 115 tac tgc cac aac cag ggt ata gtt cac agg gac ctg aca cct gac aat 919 Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu Thr Pro Asp Asn 120 125 130 att atg gta gaa aaa gat ggg aaa gtg aag atc att gat ttt gga ctc 967 Ile Met Val Glu Lys Asp Gly Lys Val Lys Ile Ile Asp Phe Gly Leu 135 140 145 ggc acc caa gag aag cca ggg caa aac cac aac tta ttc tgt gag att 1015 Gly Thr Gln Glu Lys Pro Gly Gln Asn His Asn Leu Phe Cys Glu Ile 150 155 160 tac cca ttt agt act cct gag gtg ctc ttt aac aga ccc tat gat atg 1063 Tyr Pro Phe Ser Thr Pro Glu Val Leu Phe Asn Arg Pro Tyr Asp Met 165 170 175 180 cgc aag atc gat gtg tgg ggt ctt gga gtt gtg ctg tat ttt atg gta 1111 Arg Lys Ile Asp Val Trp Gly Leu Gly Val Val Leu Tyr Phe Met Val 185 190 195 act gga aag att ctg ttt gat act gcc agc gta gaa aag ctg cga aag 1159 Thr Gly Lys Ile Leu Phe Asp Thr Ala Ser Val Glu Lys Leu Arg Lys 200 205 210 caa att gtt gca gaa aag tgt tct gtt ccc tgt aga ctg tca gta gag 1207 Gln Ile Val Ala Glu Lys Cys Ser Val Pro Cys Arg Leu Ser Val Glu 215 220 225 ctc caa gac ctg att aga ctt tta atg acg gac atc ccc gaa ctt agg 1255 Leu Gln Asp Leu Ile Arg Leu Leu Met Thr Asp Ile Pro Glu Leu Arg 230 235 240 ccc act gtt gct gaa gtt atg gtg cat ccc tgg gtc aca gaa ggc tca 1303 Pro Thr Val Ala Glu Val Met Val His Pro Trp Val Thr Glu Gly Ser 245 250 255 260 ggg gtg tta cca gat cct tgt gaa gaa cat ata ccc ctc aag cca gac 1351 Gly Val Leu Pro Asp Pro Cys Glu Glu His Ile Pro Leu Lys Pro Asp 265 270 275 cct gcg att gca aaa gca atg gga ttt atc ggg ttc caa gct caa gac 1399 Pro Ala Ile Ala Lys Ala Met Gly Phe Ile Gly Phe Gln Ala Gln Asp 280 285 290 att gaa gat tcg tta tgt cag aga aaa ttc aac gaa acc atg gca tct 1447 Ile Glu Asp Ser Leu Cys Gln Arg Lys Phe Asn Glu Thr Met Ala Ser 295 300 305 tat tgt cta ctg aaa aaa cag att ctt aag gaa tgt gac agg cca atc 1495 Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys Asp Arg Pro Ile 310 315 320 cgg gct cag ccc atg aat cca tct gtg acc cca ctc tct tcc ctt gtt 1543 Arg Ala Gln Pro Met Asn Pro Ser Val Thr Pro Leu Ser Ser Leu Val 325 330 335 340 gat gct cct act ttc cat ctc gga ctt cgg agg aca gag act gaa ccc 1591 Asp Ala Pro Thr Phe His Leu Gly Leu Arg Arg Thr Glu Thr Glu Pro 345 350 355 aca ggt ctc aga tta tct gac aat aag gaa gtg cct gtc tgt ggc aat 1639 Thr Gly Leu Arg Leu Ser Asp Asn Lys Glu Val Pro Val Cys Gly Asn 360 365 370 agt act agt aag aaa aga gag aga agt ttc agt ggg ccg ggt gtt ctc 1687 Ser Thr Ser Lys Lys Arg Glu Arg Ser Phe Ser Gly Pro Gly Val Leu 375 380 385 agc agg ccg att aac aca aca ccc aca atg gac caa aca cac acc cgt 1735 Ser Arg Pro Ile Asn Thr Thr Pro Thr Met Asp Gln Thr His Thr Arg 390 395 400 act tgg agt ggt ccc tgc att tac tca aat gtt tgc aca atc cat cca 1783 Thr Trp Ser Gly Pro Cys Ile Tyr Ser Asn Val Cys Thr Ile His Pro 405 410 415 420 aac agc atc aat gag agt aca gaa ggc cac atc agt acc tca gca gag 1831 Asn Ser Ile Asn Glu Ser Thr Glu Gly His Ile Ser Thr Ser Ala Glu 425 430 435 gat aag cct gtc cac agc aga ggc tgg ccc aga ggc atc aag ggc tgg 1879 Asp Lys Pro Val His Ser Arg Gly Trp Pro Arg Gly Ile Lys Gly Trp 440 445 450 act agg aag ata gga aat gca atg agg aag ctc tgt tgc tgt atc cca 1927 Thr Arg Lys Ile Gly Asn Ala Met Arg Lys Leu Cys Cys Cys Ile Pro 455 460 465 tcc aaa gag aca tct cac ctg ggg cag aga aga gtc tgc cca aaa att 1975 Ser Lys Glu Thr Ser His Leu Gly Gln Arg Arg Val Cys Pro Lys Ile 470 475 480 taagacacag gaaggatgtc aggagaatga gcatccagca tggcccagcc tttcagaccg 2035 aaggcaagct ctacctgatc ctggacttcc tgcggggagg tgacctcttc accaggcttt 2095 ccaaagaggt gatgttcacg gaggaggatg tcaagttcta cctggctgag ctggccttgg 2155 ctctagacca cctccatggc ctggggatca tctacaggga tctgaagcca gagaatatcc 2215 tcctggatga agagggacat attaagatca cagattttgg cttgagcaag gaggccaccg 2275 accatgacaa gagagcctat tcattttgtg ggactattga atacatggcg cccgaggtgg 2335 tgaaccggcg tggacacaca cagagtgccg actggtggtc cttcggtgtg ctcatgttcg 2395 agatgctcac agggtccctg ccattccagg ggaaggacag gaaggaaaca atggcccgca 2455 tcctcaaagc aaagctgggt atgccttagt tcctcagtgc ggaggctcag agcctgctca 2515 gggccctttt caagcggaac ccctgcaacc ggctaggtaa gggtccctgt gacaccccca 2575 ccccaggaat gcaatgaggc tgccctctag acccccctta ggaatgtgag aggccaccat 2635 tctgttcccc acgggatgtg gaggacttcc tccttatgcc ccaactctga actgtatgct 2695 tttccttgct aaggttgcag gaagcagagg taccccgacg ctggggaaac actcacatgt 2755 ggcctggcgc ccacaggcac gtggacttat caggattgct gaaaggcatt tgaaaaaaaa 2815 aaaaaaaaaa aa 2827 12 484 PRT Mus musculus 12 Met Glu Lys Phe His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln 1 5 10 15 Gly Gly Cys Ala Gln Val Lys Leu Ala Arg His Arg Leu Thr Gly Thr 20 25 30 His Val Ala Val Lys Val Ile Val Lys Arg Glu Cys Trp Phe Asn Pro 35 40 45 Val Met Ser Glu Ala Glu Leu Leu Met Met Thr Asp His Pro Asn Ile 50 55 60 Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile 65 70 75 80 Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala 85 90 95 Gly Tyr Leu Gln Glu Asp Glu Ala Arg Pro Leu Phe Lys Gln Leu Leu 100 105 110 Ser Ala Met Asn Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu 115 120 125 Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Lys Val Lys Ile Ile 130 135 140 Asp Phe Gly Leu Gly Thr Gln Glu Lys Pro Gly Gln Asn His Asn Leu 145 150 155 160 Phe Cys Glu Ile Tyr Pro Phe Ser Thr Pro Glu Val Leu Phe Asn Arg 165 170 175 Pro Tyr Asp Met Arg Lys Ile Asp Val Trp Gly Leu Gly Val Val Leu 180 185 190 Tyr Phe Met Val Thr Gly Lys Ile Leu Phe Asp Thr Ala Ser Val Glu 195 200 205 Lys Leu Arg Lys Gln Ile Val Ala Glu Lys Cys Ser Val Pro Cys Arg 210 215 220 Leu Ser Val Glu Leu Gln Asp Leu Ile Arg Leu Leu Met Thr Asp Ile 225 230 235 240 Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val His Pro Trp Val 245 250 255 Thr Glu Gly Ser Gly Val Leu Pro Asp Pro Cys Glu Glu His Ile Pro 260 265 270 Leu Lys Pro Asp Pro Ala Ile Ala Lys Ala Met Gly Phe Ile Gly Phe 275 280 285 Gln Ala Gln Asp Ile Glu Asp Ser Leu Cys Gln Arg Lys Phe Asn Glu 290 295 300 Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys 305 310 315 320 Asp Arg Pro Ile Arg Ala Gln Pro Met Asn Pro Ser Val Thr Pro Leu 325 330 335 Ser Ser Leu Val Asp Ala Pro Thr Phe His Leu Gly Leu Arg Arg Thr 340 345 350 Glu Thr Glu Pro Thr Gly Leu Arg Leu Ser Asp Asn Lys Glu Val Pro 355 360 365 Val Cys Gly Asn Ser Thr Ser Lys Lys Arg Glu Arg Ser Phe Ser Gly 370 375 380 Pro Gly Val Leu Ser Arg Pro Ile Asn Thr Thr Pro Thr Met Asp Gln 385 390 395 400 Thr His Thr Arg Thr Trp Ser Gly Pro Cys Ile Tyr Ser Asn Val Cys 405 410 415 Thr Ile His Pro Asn Ser Ile Asn Glu Ser Thr Glu Gly His Ile Ser 420 425 430 Thr Ser Ala Glu Asp Lys Pro Val His Ser Arg Gly Trp Pro Arg Gly 435 440 445 Ile Lys Gly Trp Thr Arg Lys Ile Gly Asn Ala Met Arg Lys Leu Cys 450 455 460 Cys Cys Ile Pro Ser Lys Glu Thr Ser His Leu Gly Gln Arg Arg Val 465 470 475 480 Cys Pro Lys Ile 13 1787 DNA Mus musculus CDS (95)..(1606) 13 cacgtgtggt aggtgtgaga acacctcttg tgttgaggcc gtcagtcagc ttggccattg 60 tttctaaggt agcatttata ctttgttacc tcaa atg ggg tct ggg agt caa cag 115 Met Gly Ser Gly Ser Gln Gln 1 5 aag tca gaa aag ctc aga tcc aag ccc cct ttc tct gaa atg gag aac 163 Lys Ser Glu Lys Leu Arg Ser Lys Pro Pro Phe Ser Glu Met Glu Asn 10 15 20 ttt cat gct caa tat gag atg cta ggg act att ggc cat gga ggc agc 211 Phe His Ala Gln Tyr Glu Met Leu Gly Thr Ile Gly His Gly Gly Ser 25 30 35 aca aag gtg aag ctg gcc cga cac cgc ctc aca ggc acc cac gtg gct 259 Thr Lys Val Lys Leu Ala Arg His Arg Leu Thr Gly Thr His Val Ala 40 45 50 55 gtc aaa atg att cca aag agg gag tat tgg tgc aaa cct ctc atg tct 307 Val Lys Met Ile Pro Lys Arg Glu Tyr Trp Cys Lys Pro Leu Met Ser 60 65 70 gag gca gag tta ctg atg atg gcc gat cat ccg aat atc atc tct ctc 355 Glu Ala Glu Leu Leu Met Met Ala Asp His Pro Asn Ile Ile Ser Leu 75 80 85 ctt caa gtc att gag acc aag aag aaa gta tac ctc att atg gag ttg 403 Leu Gln Val Ile Glu Thr Lys Lys Lys Val Tyr Leu Ile Met Glu Leu 90 95 100 tgt gag ggt aaa tca ctt tac caa cac atc aga aac gct ggc tac ctg 451 Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Arg Asn Ala Gly Tyr Leu 105 110 115 cag gag gat gaa gca cga gca tta ttc aag cag ctc tta agt gct ata 499 Gln Glu Asp Glu Ala Arg Ala Leu Phe Lys Gln Leu Leu Ser Ala Ile 120 125 130 135 aac tac tgc cgc aac cag ggt ata gtt cac agg gac ctg aaa ccc gac 547 Asn Tyr Cys Arg Asn Gln Gly Ile Val His Arg Asp Leu Lys Pro Asp 140 145 150 aat att atg gta gaa aaa gat ggg aga gta aag atc att gat ttt ggg 595 Asn Ile Met Val Glu Lys Asp Gly Arg Val Lys Ile Ile Asp Phe Gly 155 160 165 ctt ggc atc caa gtg aag cca ggg caa aaa cta aac tta ttc tgt ggg 643 Leu Gly Ile Gln Val Lys Pro Gly Gln Lys Leu Asn Leu Phe Cys Gly 170 175 180 act tac cca ttt agt gct cct gag gtg ctc ctt agc aga ccc tat gat 691 Thr Tyr Pro Phe Ser Ala Pro Glu Val Leu Leu Ser Arg Pro Tyr Asp 185 190 195 ggg ccc aag atc gat gta tgg act ctt gga gtt gtg cta tac ttt atg 739 Gly Pro Lys Ile Asp Val Trp Thr Leu Gly Val Val Leu Tyr Phe Met 200 205 210 215 gta act gga aag atc cca ttt gat gct gcc agc ata gaa aag ctg cgg 787 Val Thr Gly Lys Ile Pro Phe Asp Ala Ala Ser Ile Glu Lys Leu Arg 220 225 230 aag caa att gtt gca gga aag tat tct gtt ccc tgt aga ctg tca gta 835 Lys Gln Ile Val Ala Gly Lys Tyr Ser Val Pro Cys Arg Leu Ser Val 235 240 245 aag ctt cat cac ctg att act ctt tta atg aca gac aac cct gaa ctt 883 Lys Leu His His Leu Ile Thr Leu Leu Met Thr Asp Asn Pro Glu Leu 250 255 260 agg ccc act gtt gct gaa gtt atg atg cat ccc tgg gtc aca aaa ggc 931 Arg Pro Thr Val Ala Glu Val Met Met His Pro Trp Val Thr Lys Gly 265 270 275 tca ggg gtg ttc cca gat cct tgt gaa gaa cag ata ccc ctc aag cca 979 Ser Gly Val Phe Pro Asp Pro Cys Glu Glu Gln Ile Pro Leu Lys Pro 280 285 290 295 gac cct gcg att gta aaa gca atg gga cat att ggg ttc caa gct caa 1027 Asp Pro Ala Ile Val Lys Ala Met Gly His Ile Gly Phe Gln Ala Gln 300 305 310 gac att gaa gat tct tta cgt cag aga aaa ttc aac gaa acc atg gca 1075 Asp Ile Glu Asp Ser Leu Arg Gln Arg Lys Phe Asn Glu Thr Met Ala 315 320 325 tct tat tgt cta ctg aaa aaa cag ctt ctt aag gaa tgt gac agg cca 1123 Ser Tyr Cys Leu Leu Lys Lys Gln Leu Leu Lys Glu Cys Asp Arg Pro 330 335 340 atc cgg gct cag ccc atg aat cca tcg gtg acc cca ttc ccc tcc ctt 1171 Ile Arg Ala Gln Pro Met Asn Pro Ser Val Thr Pro Phe Pro Ser Leu 345 350 355 gtt gat act cct act ttc cat ctc gga ctt cgg agg aga gag act gaa 1219 Val Asp Thr Pro Thr Phe His Leu Gly Leu Arg Arg Arg Glu Thr Glu 360 365 370 375 ccc acg agt ctc aga tta tct gct aat agg caa atg tct gtc tgt gga 1267 Pro Thr Ser Leu Arg Leu Ser Ala Asn Arg Gln Met Ser Val Cys Gly 380 385 390 agg agt act agt aag aaa aga gac aga agt ttc agt tgg ccc ggt gtt 1315 Arg Ser Thr Ser Lys Lys Arg Asp Arg Ser Phe Ser Trp Pro Gly Val 395 400 405 ctc agc agg ccg att aac ata aca ccc aca atg gac caa aca cac acc 1363 Leu Ser Arg Pro Ile Asn Ile Thr Pro Thr Met Asp Gln Thr His Thr 410 415 420 tgt act agg agt gtt ccc tgc att aac tca aat ttt tgc ata atc cat 1411 Cys Thr Arg Ser Val Pro Cys Ile Asn Ser Asn Phe Cys Ile Ile His 425 430 435 cca aac agc agc gac gag agt aca gaa ggc cac acc agt gcc tca gca 1459 Pro Asn Ser Ser Asp Glu Ser Thr Glu Gly His Thr Ser Ala Ser Ala 440 445 450 455 gag gat aag cct gtc cgc agc aga ggc tgg ccc aga ggc atc aag ggc 1507 Glu Asp Lys Pro Val Arg Ser Arg Gly Trp Pro Arg Gly Ile Lys Gly 460 465 470 tgg act agc aag ata gga aat gcg atg agg aag ctc tgt tgc tgt atc 1555 Trp Thr Ser Lys Ile Gly Asn Ala Met Arg Lys Leu Cys Cys Cys Ile 475 480 485 cca tca aat gag aca tct cac ctg ggg cag agg aga gtc tcc ccc aaa 1603 Pro Ser Asn Glu Thr Ser His Leu Gly Gln Arg Arg Val Ser Pro Lys 490 495 500 aaa taagacacag gaagggtgtc aggagaacga gcattcggct cggcacagaa 1656 Lys gatcactaga ggatgccgga tgctatgatt caacagttat agtattggaa aggacccatg 1716 tatagacatg gacctgcaaa agggaacctt gtggaaaggc atcatgttct gggtccagcc 1776 agggggaaga a 1787 14 504 PRT Mus musculus 14 Met Gly Ser Gly Ser Gln Gln Lys Ser Glu Lys Leu Arg Ser Lys Pro 1 5 10 15 Pro Phe Ser Glu Met Glu Asn Phe His Ala Gln Tyr Glu Met Leu Gly 20 25 30 Thr Ile Gly His Gly Gly Ser Thr Lys Val Lys Leu Ala Arg His Arg 35 40 45 Leu Thr Gly Thr His Val Ala Val Lys Met Ile Pro Lys Arg Glu Tyr 50 55 60 Trp Cys Lys Pro Leu Met Ser Glu Ala Glu Leu Leu Met Met Ala Asp 65 70 75 80 His Pro Asn Ile Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys 85 90 95 Val Tyr Leu Ile Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His 100 105 110 Ile Arg Asn Ala Gly Tyr Leu Gln Glu Asp Glu Ala Arg Ala Leu Phe 115 120 125 Lys Gln Leu Leu Ser Ala Ile Asn Tyr Cys Arg Asn Gln Gly Ile Val 130 135 140 His Arg Asp Leu Lys Pro Asp Asn Ile Met Val Glu Lys Asp Gly Arg 145 150 155 160 Val Lys Ile Ile Asp Phe Gly Leu Gly Ile Gln Val Lys Pro Gly Gln 165 170 175 Lys Leu Asn Leu Phe Cys Gly Thr Tyr Pro Phe Ser Ala Pro Glu Val 180 185 190 Leu Leu Ser Arg Pro Tyr Asp Gly Pro Lys Ile Asp Val Trp Thr Leu 195 200 205 Gly Val Val Leu Tyr Phe Met Val Thr Gly Lys Ile Pro Phe Asp Ala 210 215 220 Ala Ser Ile Glu Lys Leu Arg Lys Gln Ile Val Ala Gly Lys Tyr Ser 225 230 235 240 Val Pro Cys Arg Leu Ser Val Lys Leu His His Leu Ile Thr Leu Leu 245 250 255 Met Thr Asp Asn Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Met 260 265 270 His Pro Trp Val Thr Lys Gly Ser Gly Val Phe Pro Asp Pro Cys Glu 275 280 285 Glu Gln Ile Pro Leu Lys Pro Asp Pro Ala Ile Val Lys Ala Met Gly 290 295 300 His Ile Gly Phe Gln Ala Gln Asp Ile Glu Asp Ser Leu Arg Gln Arg 305 310 315 320 Lys Phe Asn Glu Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Leu 325 330 335 Leu Lys Glu Cys Asp Arg Pro Ile Arg Ala Gln Pro Met Asn Pro Ser 340 345 350 Val Thr Pro Phe Pro Ser Leu Val Asp Thr Pro Thr Phe His Leu Gly 355 360 365 Leu Arg Arg Arg Glu Thr Glu Pro Thr Ser Leu Arg Leu Ser Ala Asn 370 375 380 Arg Gln Met Ser Val Cys Gly Arg Ser Thr Ser Lys Lys Arg Asp Arg 385 390 395 400 Ser Phe Ser Trp Pro Gly Val Leu Ser Arg Pro Ile Asn Ile Thr Pro 405 410 415 Thr Met Asp Gln Thr His Thr Cys Thr Arg Ser Val Pro Cys Ile Asn 420 425 430 Ser Asn Phe Cys Ile Ile His Pro Asn Ser Ser Asp Glu Ser Thr Glu 435 440 445 Gly His Thr Ser Ala Ser Ala Glu Asp Lys Pro Val Arg Ser Arg Gly 450 455 460 Trp Pro Arg Gly Ile Lys Gly Trp Thr Ser Lys Ile Gly Asn Ala Met 465 470 475 480 Arg Lys Leu Cys Cys Cys Ile Pro Ser Asn Glu Thr Ser His Leu Gly 485 490 495 Gln Arg Arg Val Ser Pro Lys Lys 500 15 2001 DNA Mus musculus CDS (343)..(1641) 15 gtgtgggagg agcttgtgtg tgtgagttgt gttttaagtt tatttgcgtc tcgtgagtac 60 ctttgggttt gtgtgtgtgt gtctgtgtgt gtttgtgtgt gtataactgt gggtgactgt 120 taagagcacc tgtgtgtttg tacgtgagtg tgtaagactg tgtgtgtgca caagagcgtg 180 tgtaggtgca catgttgtag gtgtgagaac acctgttgtg tttaggccat cagtcagctt 240 ggccattgtt tctaaggtag catttatact ttgttacctc aagtgggctc tgggagtcaa 300 gagaaatcag aaaagctcag atccaagccc cctttctctg ac atg gag aaa ttt 354 Met Glu Lys Phe 1 cat gct caa tat gaa atg cta gag act atc ggc cag gga ggc tgc gcc 402 His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln Gly Gly Cys Ala 5 10 15 20 cag gtg aag ctg gcc cag cac cgc ctc aca ggc acc cac gtg gct gtc 450 Gln Val Lys Leu Ala Gln His Arg Leu Thr Gly Thr His Val Ala Val 25 30 35 aaa gtg att gta aag agg gag tgt tgg ttc aac cct gtc atg tct gag 498 Lys Val Ile Val Lys Arg Glu Cys Trp Phe Asn Pro Val Met Ser Glu 40 45 50 gca gag tta ctg atg atg acc gat cat ccg aat atc atc tct ctc ctt 546 Ala Glu Leu Leu Met Met Thr Asp His Pro Asn Ile Ile Ser Leu Leu 55 60 65 caa gtc atc gag acc aag aag aaa tta tac ctc att atg gag ttg tgc 594 Gln Val Ile Glu Thr Lys Lys Lys Leu Tyr Leu Ile Met Glu Leu Cys 70 75 80 gag ggt aaa tca ctt tac caa cac atc caa aat gct ggc tac ctg cag 642 Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala Gly Tyr Leu Gln 85 90 95 100 gag gat gaa gca tgc cca tta ttc aag cag ctc tta agt gct gtg aac 690 Glu Asp Glu Ala Cys Pro Leu Phe Lys Gln Leu Leu Ser Ala Val Asn 105 110 115 tac tgc cac aac cag ggt ata gtt cac agg gac ctg aca cct gac aat 738 Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu Thr Pro Asp Asn 120 125 130 att atg gta gaa aaa gat ggg aaa gtg aag atc att gat ttt gga ctc 786 Ile Met Val Glu Lys Asp Gly Lys Val Lys Ile Ile Asp Phe Gly Leu 135 140 145 ggc acc caa gag aag cca gcg caa aaa ctc aac tta ttc tgt gag aat 834 Gly Thr Gln Glu Lys Pro Ala Gln Lys Leu Asn Leu Phe Cys Glu Asn 150 155 160 tac cca ttt agt acc cct gag gtg ctc ctt agc aga ccc tat gat atg 882 Tyr Pro Phe Ser Thr Pro Glu Val Leu Leu Ser Arg Pro Tyr Asp Met 165 170 175 180 cgc aag atc gat gtg tgg ggt ctt gga gtt gtg ctg tat ttt atg gta 930 Arg Lys Ile Asp Val Trp Gly Leu Gly Val Val Leu Tyr Phe Met Val 185 190 195 act gga aag att ctg ttt gat gct gcc agc ata gaa aag ctg cga aag 978 Thr Gly Lys Ile Leu Phe Asp Ala Ala Ser Ile Glu Lys Leu Arg Lys 200 205 210 caa att gtt gca gga aag tgt tct gtt ccc tgt aga ctg tca gta gag 1026 Gln Ile Val Ala Gly Lys Cys Ser Val Pro Cys Arg Leu Ser Val Glu 215 220 225 ctc caa gac ctg att aga ctt tta atg acg gac aac ccc gaa ctt agg 1074 Leu Gln Asp Leu Ile Arg Leu Leu Met Thr Asp Asn Pro Glu Leu Arg 230 235 240 ccc act gtt gct gaa gtt atg gtg cat ccc tgg gtc aca gta ggc tca 1122 Pro Thr Val Ala Glu Val Met Val His Pro Trp Val Thr Val Gly Ser 245 250 255 260 ggg gtg ttc cca gat cct tgt gaa gaa cag ata tcc ctc aag cca gac 1170 Gly Val Phe Pro Asp Pro Cys Glu Glu Gln Ile Ser Leu Lys Pro Asp 265 270 275 cct gcg att gta aaa gca atg gga tat atc ggg ttc cga gct caa gaa 1218 Pro Ala Ile Val Lys Ala Met Gly Tyr Ile Gly Phe Arg Ala Gln Glu 280 285 290 att gaa gat tcg tta cgt cag aga aaa ttc aac gaa acc atg gca tct 1266 Ile Glu Asp Ser Leu Arg Gln Arg Lys Phe Asn Glu Thr Met Ala Ser 295 300 305 tat tgt cta ctg aaa aaa cag att ctt aag gaa tgt gac agg cca atc 1314 Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys Asp Arg Pro Ile 310 315 320 cgg gct cag ccc atg aat cca tcg ctg acc cca ttc cct tcc ctt gtt 1362 Arg Ala Gln Pro Met Asn Pro Ser Leu Thr Pro Phe Pro Ser Leu Val 325 330 335 340 gat act cct act tcc cat ctc gga ctt cgg agg aga gag act gaa ccc 1410 Asp Thr Pro Thr Ser His Leu Gly Leu Arg Arg Arg Glu Thr Glu Pro 345 350 355 aca ggt ctc agc tta tct gcc aat agg caa gtg tct gtc tgt ggc aag 1458 Thr Gly Leu Ser Leu Ser Ala Asn Arg Gln Val Ser Val Cys Gly Lys 360 365 370 agt act agt aag aaa aga gac aga agt ttc agt tgg ccc ggt gtt cta 1506 Ser Thr Ser Lys Lys Arg Asp Arg Ser Phe Ser Trp Pro Gly Val Leu 375 380 385 ggc agg ccg atc cac aca aca ccc aca atg gac caa aca cac acc cgt 1554 Gly Arg Pro Ile His Thr Thr Pro Thr Met Asp Gln Thr His Thr Arg 390 395 400 act agg agt gtt ccc tgc att tac tca aat ttt tgc aca atc cat cca 1602 Thr Arg Ser Val Pro Cys Ile Tyr Ser Asn Phe Cys Thr Ile His Pro 405 410 415 420 aac agc atc gat gag agt aca gaa ggc cac acc agt gcc taagcagagg 1651 Asn Ser Ile Asp Glu Ser Thr Glu Gly His Thr Ser Ala 425 430 ataagcctgt ccgcagcaga ggctggccca gaggcatcaa gggctggact aggaagatag 1711 gaaatgcgat gaggaagctc tgttgctgta tcccatcaaa agagacatct cacctggggc 1771 agagcaaagt ctccccaaaa aaataagaca caggaagggt gtcaggagaa agagcatctg 1831 gcacggccca gaagatcacc agaggatgcc ggatgctatg attcgacagt tataatattg 1891 gaaaggaccc atgtatagac attgtcctgc aaaagggaac cttgtggaaa ggcatcatgt 1951 tctgggttca gcgtgcttca ctcagagccc cgggtccagc cagggggaag 2001 16 433 PRT Mus musculus 16 Met Glu Lys Phe His Ala Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln 1 5 10 15 Gly Gly Cys Ala Gln Val Lys Leu Ala Gln His Arg Leu Thr Gly Thr 20 25 30 His Val Ala Val Lys Val Ile Val Lys Arg Glu Cys Trp Phe Asn Pro 35 40 45 Val Met Ser Glu Ala Glu Leu Leu Met Met Thr Asp His Pro Asn Ile 50 55 60 Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys Leu Tyr Leu Ile 65 70 75 80 Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala 85 90 95 Gly Tyr Leu Gln Glu Asp Glu Ala Cys Pro Leu Phe Lys Gln Leu Leu 100 105 110 Ser Ala Val Asn Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu 115 120 125 Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Lys Val Lys Ile Ile 130 135 140 Asp Phe Gly Leu Gly Thr Gln Glu Lys Pro Ala Gln Lys Leu Asn Leu 145 150 155 160 Phe Cys Glu Asn Tyr Pro Phe Ser Thr Pro Glu Val Leu Leu Ser Arg 165 170 175 Pro Tyr Asp Met Arg Lys Ile Asp Val Trp Gly Leu Gly Val Val Leu 180 185 190 Tyr Phe Met Val Thr Gly Lys Ile Leu Phe Asp Ala Ala Ser Ile Glu 195 200 205 Lys Leu Arg Lys Gln Ile Val Ala Gly Lys Cys Ser Val Pro Cys Arg 210 215 220 Leu Ser Val Glu Leu Gln Asp Leu Ile Arg Leu Leu Met Thr Asp Asn 225 230 235 240 Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val His Pro Trp Val 245 250 255 Thr Val Gly Ser Gly Val Phe Pro Asp Pro Cys Glu Glu Gln Ile Ser 260 265 270 Leu Lys Pro Asp Pro Ala Ile Val Lys Ala Met Gly Tyr Ile Gly Phe 275 280 285 Arg Ala Gln Glu Ile Glu Asp Ser Leu Arg Gln Arg Lys Phe Asn Glu 290 295 300 Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys 305 310 315 320 Asp Arg Pro Ile Arg Ala Gln Pro Met Asn Pro Ser Leu Thr Pro Phe 325 330 335 Pro Ser Leu Val Asp Thr Pro Thr Ser His Leu Gly Leu Arg Arg Arg 340 345 350 Glu Thr Glu Pro Thr Gly Leu Ser Leu Ser Ala Asn Arg Gln Val Ser 355 360 365 Val Cys Gly Lys Ser Thr Ser Lys Lys Arg Asp Arg Ser Phe Ser Trp 370 375 380 Pro Gly Val Leu Gly Arg Pro Ile His Thr Thr Pro Thr Met Asp Gln 385 390 395 400 Thr His Thr Arg Thr Arg Ser Val Pro Cys Ile Tyr Ser Asn Phe Cys 405 410 415 Thr Ile His Pro Asn Ser Ile Asp Glu Ser Thr Glu Gly His Thr Ser 420 425 430 Ala 17 1591 DNA Mus musculus CDS (92)..(1390) 17 cttaggtagc atttatactt tgttacctca agtgggctct gggagtcaag cgaagtcaga 60 aaagctcaga tccaagcccc ctttctctga c atg gag aaa ttt cat tct caa 112 Met Glu Lys Phe His Ser Gln 1 5 tat gag atg cta gag act atc ggc cag gga agc tgc gcc cag gtg aag 160 Tyr Glu Met Leu Glu Thr Ile Gly Gln Gly Ser Cys Ala Gln Val Lys 10 15 20 ctg gcc cag cac cgc ctc aca ggc acc cac gtg gct gtc aaa gtg att 208 Leu Ala Gln His Arg Leu Thr Gly Thr His Val Ala Val Lys Val Ile 25 30 35 gta aag agg gag tgt tgg ttc aac cct gtc atg tct gag gca gag tta 256 Val Lys Arg Glu Cys Trp Phe Asn Pro Val Met Ser Glu Ala Glu Leu 40 45 50 55 ctg atg atg acc gat cat ccg aat atc atc tct ctc ctt caa gtc atc 304 Leu Met Met Thr Asp His Pro Asn Ile Ile Ser Leu Leu Gln Val Ile 60 65 70 gag acc aag aag aaa tta tac ctc att atg gag ttg tgc gag ggt aaa 352 Glu Thr Lys Lys Lys Leu Tyr Leu Ile Met Glu Leu Cys Glu Gly Lys 75 80 85 tca ctt tac caa cac atc caa aat gct ggc tac ctg cag gag gat gaa 400 Ser Leu Tyr Gln His Ile Gln Asn Ala Gly Tyr Leu Gln Glu Asp Glu 90 95 100 gca tgc cca tta ttc aag cag ctc tta agt gct gtg aac tac tgc cac 448 Ala Cys Pro Leu Phe Lys Gln Leu Leu Ser Ala Val Asn Tyr Cys His 105 110 115 aac cag ggt ata gtt cac agg gac ctg aca cct gac aat att atg gta 496 Asn Gln Gly Ile Val His Arg Asp Leu Thr Pro Asp Asn Ile Met Val 120 125 130 135 gaa aaa gat ggg aaa gtg aag atc att gat ttt gga ctc ggc acc caa 544 Glu Lys Asp Gly Lys Val Lys Ile Ile Asp Phe Gly Leu Gly Thr Gln 140 145 150 gag aag cca gcg caa aaa ctc aac tta ttc tgt gag aat tac cca ttt 592 Glu Lys Pro Ala Gln Lys Leu Asn Leu Phe Cys Glu Asn Tyr Pro Phe 155 160 165 agt acc cct gag gtg ctc ctt agc aga ccc tat gat atg cgc aag atc 640 Ser Thr Pro Glu Val Leu Leu Ser Arg Pro Tyr Asp Met Arg Lys Ile 170 175 180 gat gtg tgg ggt ctt gga gtt gtg ctg tat ttt atg gta act gga aag 688 Asp Val Trp Gly Leu Gly Val Val Leu Tyr Phe Met Val Thr Gly Lys 185 190 195 att ctg ttt gat gct gcc agc ata gaa aag ctg cga aag caa att gtt 736 Ile Leu Phe Asp Ala Ala Ser Ile Glu Lys Leu Arg Lys Gln Ile Val 200 205 210 215 gca gga aag tgt tct gtt ccc tgt aga ctg tca gta gag ctc caa gac 784 Ala Gly Lys Cys Ser Val Pro Cys Arg Leu Ser Val Glu Leu Gln Asp 220 225 230 ctg att aga ctt tta atg acg gac aac ccc gaa ctt agg ccc act gtt 832 Leu Ile Arg Leu Leu Met Thr Asp Asn Pro Glu Leu Arg Pro Thr Val 235 240 245 gct gaa gtt atg gtg cat ccc tgg gtc aca gaa ggc tca ggg gtg ttc 880 Ala Glu Val Met Val His Pro Trp Val Thr Glu Gly Ser Gly Val Phe 250 255 260 cca gat cct tgt gaa gaa cag ata tcc ctc aag cca gac cct gcg att 928 Pro Asp Pro Cys Glu Glu Gln Ile Ser Leu Lys Pro Asp Pro Ala Ile 265 270 275 gta aaa gca atg gga tat atc ggg ttc cga gct caa gaa att gaa gat 976 Val Lys Ala Met Gly Tyr Ile Gly Phe Arg Ala Gln Glu Ile Glu Asp 280 285 290 295 tcg tta cgt cag aga aaa ttc aac gaa acc atg gca tct tat tgt cta 1024 Ser Leu Arg Gln Arg Lys Phe Asn Glu Thr Met Ala Ser Tyr Cys Leu 300 305 310 ctg aaa aaa cag att ctt aag gaa tgt gac agg cca atc cgg gct cag 1072 Leu Lys Lys Gln Ile Leu Lys Glu Cys Asp Arg Pro Ile Arg Ala Gln 315 320 325 ccc atg aat cca tcg ctg acc cca ttc cct tcc ctt gtt gat act cct 1120 Pro Met Asn Pro Ser Leu Thr Pro Phe Pro Ser Leu Val Asp Thr Pro 330 335 340 act tcc cat ctc gga ctt cgg agg aga gag act gaa ccc aca ggt ctc 1168 Thr Ser His Leu Gly Leu Arg Arg Arg Glu Thr Glu Pro Thr Gly Leu 345 350 355 agc tta tct gcc aat agg caa gtg tct gtc tgt ggc aag agt act agt 1216 Ser Leu Ser Ala Asn Arg Gln Val Ser Val Cys Gly Lys Ser Thr Ser 360 365 370 375 aag aaa aga gac aga agt ttc agt tgg ccc ggt gtt cta ggc agg ccg 1264 Lys Lys Arg Asp Arg Ser Phe Ser Trp Pro Gly Val Leu Gly Arg Pro 380 385 390 atc cac aca aca ccc aca atg gac caa aca cac acc cgt act agg agt 1312 Ile His Thr Thr Pro Thr Met Asp Gln Thr His Thr Arg Thr Arg Ser 395 400 405 gtt ccc tgc att tac tca aat ttt tgc aca atc cat cca aac agc atc 1360 Val Pro Cys Ile Tyr Ser Asn Phe Cys Thr Ile His Pro Asn Ser Ile 410 415 420 gat gag agt aca gaa ggc cac acc agt gcc taagcagagg ataagcctgt 1410 Asp Glu Ser Thr Glu Gly His Thr Ser Ala 425 430 ccgcagcaga ggctggccca gaggcatcaa gggctggact aggaagatag gaaatgcgat 1470 gaggaagctc tgttgctgta tcccatcaaa agagacatct cacctggggc agagcagagt 1530 gtccccaaaa aaataagaca caggaagggt gtcaggagaa cgagcatgcg gcacggccca 1590 g 1591 18 433 PRT Mus musculus 18 Met Glu Lys Phe His Ser Gln Tyr Glu Met Leu Glu Thr Ile Gly Gln 1 5 10 15 Gly Ser Cys Ala Gln Val Lys Leu Ala Gln His Arg Leu Thr Gly Thr 20 25 30 His Val Ala Val Lys Val Ile Val Lys Arg Glu Cys Trp Phe Asn Pro 35 40 45 Val Met Ser Glu Ala Glu Leu Leu Met Met Thr Asp His Pro Asn Ile 50 55 60 Ile Ser Leu Leu Gln Val Ile Glu Thr Lys Lys Lys Leu Tyr Leu Ile 65 70 75 80 Met Glu Leu Cys Glu Gly Lys Ser Leu Tyr Gln His Ile Gln Asn Ala 85 90 95 Gly Tyr Leu Gln Glu Asp Glu Ala Cys Pro Leu Phe Lys Gln Leu Leu 100 105 110 Ser Ala Val Asn Tyr Cys His Asn Gln Gly Ile Val His Arg Asp Leu 115 120 125 Thr Pro Asp Asn Ile Met Val Glu Lys Asp Gly Lys Val Lys Ile Ile 130 135 140 Asp Phe Gly Leu Gly Thr Gln Glu Lys Pro Ala Gln Lys Leu Asn Leu 145 150 155 160 Phe Cys Glu Asn Tyr Pro Phe Ser Thr Pro Glu Val Leu Leu Ser Arg 165 170 175 Pro Tyr Asp Met Arg Lys Ile Asp Val Trp Gly Leu Gly Val Val Leu 180 185 190 Tyr Phe Met Val Thr Gly Lys Ile Leu Phe Asp Ala Ala Ser Ile Glu 195 200 205 Lys Leu Arg Lys Gln Ile Val Ala Gly Lys Cys Ser Val Pro Cys Arg 210 215 220 Leu Ser Val Glu Leu Gln Asp Leu Ile Arg Leu Leu Met Thr Asp Asn 225 230 235 240 Pro Glu Leu Arg Pro Thr Val Ala Glu Val Met Val His Pro Trp Val 245 250 255 Thr Glu Gly Ser Gly Val Phe Pro Asp Pro Cys Glu Glu Gln Ile Ser 260 265 270 Leu Lys Pro Asp Pro Ala Ile Val Lys Ala Met Gly Tyr Ile Gly Phe 275 280 285 Arg Ala Gln Glu Ile Glu Asp Ser Leu Arg Gln Arg Lys Phe Asn Glu 290 295 300 Thr Met Ala Ser Tyr Cys Leu Leu Lys Lys Gln Ile Leu Lys Glu Cys 305 310 315 320 Asp Arg Pro Ile Arg Ala Gln Pro Met Asn Pro Ser Leu Thr Pro Phe 325 330 335 Pro Ser Leu Val Asp Thr Pro Thr Ser His Leu Gly Leu Arg Arg Arg 340 345 350 Glu Thr Glu Pro Thr Gly Leu Ser Leu Ser Ala Asn Arg Gln Val Ser 355 360 365 Val Cys Gly Lys Ser Thr Ser Lys Lys Arg Asp Arg Ser Phe Ser Trp 370 375 380 Pro Gly Val Leu Gly Arg Pro Ile His Thr Thr Pro Thr Met Asp Gln 385 390 395 400 Thr His Thr Arg Thr Arg Ser Val Pro Cys Ile Tyr Ser Asn Phe Cys 405 410 415 Thr Ile His Pro Asn Ser Ile Asp Glu Ser Thr Glu Gly His Thr Ser 420 425 430 Ala 19 3641 DNA Mus musculus 19 gtacattctg tatttgaatg tatctatgtt actcatgtct gtgtcaactg gcagattata 60 cttatgtata tgtatatgta tatgtatatg tatatgtata tgtatatgta tatgtatatg 120 tatatgtata tggtgcaatg catggggaag ctaggtctag acaccttggg aaaatagtta 180 aattgaacct gccaacagat ccagcatccc agaaggtatc tcctgtgtgt atcctgcaca 240 ttgaacaagg aggagaactg accatgctag ggagaggaag tgggagaagg aagaggagga 300 gatgctgagg gaggagaggg tggtatgtgg tggaagctag gagaagaggg gaagaggttc 360 agacaggagg aggcaacttg ggggagcagt gtgaaacagg gtaaccccag ctggagagat 420 gccctgtgca gctgaggttc tcagagtccc tctcacgtgt gctttggcat tttagaagat 480 caccagagga tgccggatgc tacgattcaa cagttataat attggaaagg acccatgtat 540 agacatggac ctgcaaaagg gaaccttgtg gaaaggcatc atgttctggg ttcagcatgt 600 ttcactcaga gccccgggtc cagccagggg gaagaaagca aatgatgaaa tcccagatgg 660 tgtctgggat caccattcag agcaggggct gaaagcctgt ccaaagctgg tagagacaga 720 agcccctctg cctacccagg gtcataatca gactcctgct ctgagaataa aatagatgtt 780 tgtgaaagat gacctcggag gttttcctgc ctcttcttta cataggaaaa acgttcctgt 840 ggtgttcaaa atccccaggt agaacaactt cacaccccaa accaccagat cattaaaagt 900 gcctttgaag ggaccaagct ttcaggtacc tgaagtgggg gagtgtagaa gctgggtgtg 960 gactctgggt ccttctggac ccagcagcat gatcagaagg accagagaga acattagata 1020 caatcagctt gacccacttc cacagaggca agcccggtca ccctgtgaat tcctgcagat 1080 gtggcatgtg ttgcatccca gggtctctgc ctatgtaaga tcagagagcc tggagttagc 1140 taaatatcag tgtcccttgg cctcaaggga gaagggaggt tggattccag ccctagcatg 1200 gtcctctaat aagcagtccc cctcaaatgc agacagcaag gtctacatga tgttcacagc 1260 tcccctggcc taaaaccatc ctgtgattga tactacaaac caggaagcag ggacttgaag 1320 ttgagatcac tgactcaggc tagggagggc tccagggcac ctgatctcaa ctacaatatc 1380 agaagctgag ccacaatgac cagtggtggc aggttttctt ttctgctctc aggcccggca 1440 atgaagtcca catatgaggc tctttcctcc cagtcgtact gtctgcagat atgaggttcc 1500 tcaacagtgg attcaaaact ccagaaagga aagagctaca cattgtactc tgaaaagcag 1560 aggcccattc agggtttgag caaatcatcg ctcaatagtt agattccggg tacactatgt 1620 gctcaggagt aacacagcag catgggttct gtgagctgaa tgtggttcaa agtctgtttc 1680 aagtgtgtca gcagcacagc taatctgttc acggtgtcca cagagcttct ggtcctcgaa 1740 tgtgcctgct ccacctttgg accttagagt gtaaagtgag ccctacacgc agcacggact 1800 tggttttcta tacatcatgc caacctctgt gtttgatgac ggggcgggag tggggggtat 1860 gtggtgggag aggtgagaga aaggagagag agagagtcgg gtagagaaag ggaaggaagg 1920 agggagggga gaggtaaaag gaaaagcttc tatgtacatg gtcatggata tgtcccacca 1980 tgtgtgtgga ggttagagga catttttctc agatttacct tctactttgt ttgaaactag 2040 gtgtgtggtt tgagactaca tatgccaagg tgcctgcccc acaagctccc agacattttc 2100 ctgtctctaa tgctttccct gcttcctagg agctctgata ttgcaagtgt gtgctcagtg 2160 tccacatgca ttcaatctca ggccctccct ctttgcaggg caggtgttct aaccacttgt 2220 ctatccccta aggcccctcc catgtttttg atgagaatcc aaaaccttgg aaattatgag 2280 aaacacctct ttctgtcatc ctcacaggtg gtaataagct gccctattat atttcataag 2340 cagagttggg gtccaggaat caccccacaa accactcagc catctaagtc aagcagggat 2400 agtttattga acatataccc tgggactgat tgatcaggga tgcagatcag actcagaagt 2460 ttagactgca accctgtttc ccaagggttg cttataaaag gcaaaaacca caggagctca 2520 cggcaaccat aaaagctcac acacaggtgc aggaagtctt gccaggcagt tgggtggctg 2580 gttcgagtcc aaccttattt ttgctaactg tacaaagcaa ttccaactga ctttagttat 2640 tatgattggc cctaaacgag ggcaagggtc gggggtgttt gcaagaacac caaagcataa 2700 agcttaatgg gatatgcagt taatggttag ctgggcatga gaaaggtcct ctgtaataat 2760 ttaagatggc aggctacagg tataaaatga aatggctaca gtaatgtcag aaaggcagca 2820 gccacctacg tcttaatgag taggaccttt ttatttattt atttatttat ttatttattt 2880 atttatttaa tgttaagtgg tggcatcatc ctggacccat cagttggaat gcaaaggtga 2940 cacacagagt gtagacatga ggactttaaa gcaggaggca cagcaaacat tcaaaccaga 3000 gacctaagga catcagcatg gcctagaggt tttgatttct aaaagcctaa tgtcagtctc 3060 catagcccac ttaagccaga gccttgagtc cctcctagcc ctgccaggac aggtcctgat 3120 atgaccacat gaggagtgac tatgatgcgg cccagccagc aggtttaagc tgtggccaca 3180 cctagatttc tttgagtgtg ttgagaggag ttggtggagt tggtggagtt tggtggattt 3240 ggtggagttg gtggtgccct ttgcgatttc gttgtatcta gtgagccgtg tgtggatttt 3300 gtgtttgatt ggttcgtgtg tgagcttttg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg 3360 tgtgtgtgtg tgtgtgtgta gatcagtgtg tgtttgggag gagcttgtgt gtgtgagttg 3420 tgttttaagt ttatttgcgt gtgagtacct ttgggttttt gtgtgtgtct gtgtgtgttt 3480 gtgtgtgtat aactgtgggt gactgtaagt gcacctgtgt gtttgtacgt gagtgtgtaa 3540 gactgtgtgt gtgcacaaga gcgtgtgtag gtgcacgtgt tgtaggtgtg agaacacctg 3600 ttgtgtttag gccatcagtc agcttggtca ttgtttctaa g 3641 20 2827 DNA Mus musculus CDS (2002)..(2481) 20 gagaggagtt ggtggagttg gtggagtttg gtggatttgg tggagttggt ggtgcccttt 60 gcgatttcgt tgtatctagt gagccgtgtg tggattttgt gtttgattgg ttcgtgtgtg 120 agcttttgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtagatc 180 agtgtgtgtt tgggaggagc ttgtgtgtgt gagttgtgtt ttaagtttat ttgcgtgtga 240 gtacctttgg gtttttgtgt gtgtctgtgt gtgtttgtgt gtgtataact gtgggtgact 300 gtaagtgcac ctgtgtgttt gtacgtgagt gtgtaagact gtgtgtgtgc acaagagcgt 360 gtgtaggtgc acgtgttgta ggtgtgagaa cacctgttgt gtttaggcca tcagtcagct 420 tggtcattgt ttctaaggta gcatttatac tttgttacct caagtgggct ctgggagtca 480 acagaagtca gaaaagctca gatccaagcc ccctttttct gacatggaga aatttcatgc 540 tcaatatgag atgctagaga ctattggcca gggaggctgc gcccaggtga agctggcccg 600 acaccgcctc acaggcaccc acgtggctgt caaagtgatt gtaaagaggg agtgttggtt 660 caaccctgtc atgtctgagg cagagttact gatgatgacc gatcatccga atatcatctc 720 tctccttcaa gtcattgaga ccaagaagaa agtatacctc attatggagt tgtgcgaggg 780 taaatcactt taccaacaca tccaaaatgc tggctacctg caggaggatg aagcacgccc 840 attattcaag cagctcttaa gtgctatgaa ctactgccac aaccagggta tagttcacag 900 ggacctgaca cctgacaata ttatggtaga aaaagatggg aaagtgaaga tcattgattt 960 tggactcggc acccaagaga agccagggca aaaccacaac ttattctgtg agatttaccc 1020 atttagtact cctgaggtgc tctttaacag accctatgat atgcgcaaga tcgatgtgtg 1080 gggtcttgga gttgtgctgt attttatggt aactggaaag attctgtttg atactgccag 1140 cgtagaaaag ctgcgaaagc aaattgttgc agaaaagtgt tctgttccct gtagactgtc 1200 agtagagctc caagacctga ttagactttt aatgacggac atccccgaac ttaggcccac 1260 tgttgctgaa gttatggtgc atccctgggt cacagaaggc tcaggggtgt taccagatcc 1320 ttgtgaagaa catatacccc tcaagccaga ccctgcgatt gcaaaagcaa tgggatttat 1380 cgggttccaa gctcaagaca ttgaagattc gttatgtcag agaaaattca acgaaaccat 1440 ggcatcttat tgtctactga aaaaacagat tcttaaggaa tgtgacaggc caatccgggc 1500 tcagcccatg aatccatctg tgaccccact ctcttccctt gttgatgctc ctactttcca 1560 tctcggactt cggaggacag agactgaacc cacaggtctc agattatctg acaataagga 1620 agtgcctgtc tgtggcaata gtactagtaa gaaaagagag agaagtttca gtgggccggg 1680 tgttctcagc aggccgatta acacaacacc cacaatggac caaacacaca cccgtacttg 1740 gagtggtccc tgcatttact caaatgtttg cacaatccat ccaaacagca tcaatgagag 1800 tacagaaggc cacatcagta cctcagcaga ggataagcct gtccacagca gaggctggcc 1860 cagaggcatc aagggctgga ctaggaagat aggaaatgca atgaggaagc tctgttgctg 1920 tatcccatcc aaagagacat ctcacctggg gcagagaaga gtctgcccaa aaatttaaga 1980 cacaggaagg atgtcaggag a atg agc atc cag cat ggc cca gcc ttt cag 2031 Met Ser Ile Gln His Gly Pro Ala Phe Gln 1 5 10 acc gaa ggc aag ctc tac ctg atc ctg gac ttc ctg cgg gga ggt gac 2079 Thr Glu Gly Lys Leu Tyr Leu Ile Leu Asp Phe Leu Arg Gly Gly Asp 15 20 25 ctc ttc acc agg ctt tcc aaa gag gtg atg ttc acg gag gag gat gtc 2127 Leu Phe Thr Arg Leu Ser Lys Glu Val Met Phe Thr Glu Glu Asp Val 30 35 40 aag ttc tac ctg gct gag ctg gcc ttg gct cta gac cac ctc cat ggc 2175 Lys Phe Tyr Leu Ala Glu Leu Ala Leu Ala Leu Asp His Leu His Gly 45 50 55 ctg ggg atc atc tac agg gat ctg aag cca gag aat atc ctc ctg gat 2223 Leu Gly Ile Ile Tyr Arg Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp 60 65 70 gaa gag gga cat att aag atc aca gat ttt ggc ttg agc aag gag gcc 2271 Glu Glu Gly His Ile Lys Ile Thr Asp Phe Gly Leu Ser Lys Glu Ala 75 80 85 90 acc gac cat gac aag aga gcc tat tca ttt tgt ggg act att gaa tac 2319 Thr Asp His Asp Lys Arg Ala Tyr Ser Phe Cys Gly Thr Ile Glu Tyr 95 100 105 atg gcg ccc gag gtg gtg aac cgg cgt gga cac aca cag agt gcc gac 2367 Met Ala Pro Glu Val Val Asn Arg Arg Gly His Thr Gln Ser Ala Asp 110 115 120 tgg tgg tcc ttc ggt gtg ctc atg ttc gag atg ctc aca ggg tcc ctg 2415 Trp Trp Ser Phe Gly Val Leu Met Phe Glu Met Leu Thr Gly Ser Leu 125 130 135 cca ttc cag ggg aag gac agg aag gaa aca atg gcc cgc atc ctc aaa 2463 Pro Phe Gln Gly Lys Asp Arg Lys Glu Thr Met Ala Arg Ile Leu Lys 140 145 150 gca aag ctg ggt atg cct tagttcctca gtgcggaggc tcagagcctg 2511 Ala Lys Leu Gly Met Pro 155 160 ctcagggccc ttttcaagcg gaacccctgc aaccggctag gtaagggtcc ctgtgacacc 2571 cccaccccag gaatgcaatg aggctgccct ctagaccccc cttaggaatg tgagaggcca 2631 ccattctgtt ccccacggga tgtggaggac ttcctcctta tgccccaact ctgaactgta 2691 tgcttttcct tgctaaggtt gcaggaagca gaggtacccc gacgctgggg aaacactcac 2751 atgtggcctg gcgcccacag gcacgtggac ttatcaggat tgctgaaagg catttgaaaa 2811 aaaaaaaaaa aaaaaa 2827 21 160 PRT Mus musculus 21 Met Ser Ile Gln His Gly Pro Ala Phe Gln Thr Glu Gly Lys Leu Tyr 1 5 10 15 Leu Ile Leu Asp Phe Leu Arg Gly Gly Asp Leu Phe Thr Arg Leu Ser 20 25 30 Lys Glu Val Met Phe Thr Glu Glu Asp Val Lys Phe Tyr Leu Ala Glu 35 40 45 Leu Ala Leu Ala Leu Asp His Leu His Gly Leu Gly Ile Ile Tyr Arg 50 55 60 Asp Leu Lys Pro Glu Asn Ile Leu Leu Asp Glu Glu Gly His Ile Lys 65 70 75 80 Ile Thr Asp Phe Gly Leu Ser Lys Glu Ala Thr Asp His Asp Lys Arg 85 90 95 Ala Tyr Ser Phe Cys Gly Thr Ile Glu Tyr Met Ala Pro Glu Val Val 100 105 110 Asn Arg Arg Gly His Thr Gln Ser Ala Asp Trp Trp Ser Phe Gly Val 115 120 125 Leu Met Phe Glu Met Leu Thr Gly Ser Leu Pro Phe Gln Gly Lys Asp 130 135 140 Arg Lys Glu Thr Met Ala Arg Ile Leu Lys Ala Lys Leu Gly Met Pro 145 150 155 160 22 17 DNA Artificial Sequence Primer 22 atgggcagag caatggt 17 23 17 DNA Artificial Sequence Primer 23 caggttcagg gggaggt 17 24 17 DNA Artificial Sequence Primer 24 cttccccctg gctggac 17 25 15 DNA Artificial Sequence Primer 25 gccaaccagg ggata 15 26 18 DNA Artificial Sequence Primer 26 ctgtccggtc atactctt 18 27 18 DNA Artificial Sequence Primer 27 cttgtgtcct tgggagaa 18 28 17 DNA Artificial Sequence Primer 28 ggtgccatcc acttcac 17 29 20 DNA Artificial Sequence Primer 29 cgcagcaaaa gcaggagcag 20 30 20 DNA Artificial Sequence Primer 30 catcggacgg tggcattttt 20 31 20 DNA Artificial Sequence Primer 31 tgctcaagcc aaaatctgtg 20 32 20 DNA Artificial Sequence Primer 32 atggcctggg gatcatctac 20 33 20 DNA Artificial Sequence Primer 33 caccgcttgc acactgagta 20 34 18 DNA Artificial Sequence Primer 34 atcgatgtgt ggggtctt 18 35 18 DNA Artificial Sequence Primer 35 gtttgggagg agcttgtg 18 36 18 DNA Artificial Sequence Primer 36 ctagtccagc ccttgatg 18 37 18 DNA Artificial Sequence Primer 37 tggcatctta ttgtctac 18 38 18 DNA Artificial Sequence Primer 38 ccaagcccct ttttctga 18 39 24 DNA Artificial Sequence Primer 39 atttaggtga cactatagaa ggta 24 40 15 DNA Artificial Sequence Primer 40 ccccctttat ctgac 15 41 18 DNA Artificial Sequence Primer 41 tatgctggca gcatcaaa 18 42 18 DNA Artificial Sequence Primer 42 atgtaagtgg catggagt 18 43 17 DNA Artificial Sequence Primer 43 gcacaccgaa aataaaa 17 44 42 DNA Artificial Sequence Primer 44 ggcgtagtct gggacgtcgt atgggtacat gtcagaaaaa gg 42 45 45 DNA Artificial Sequence Primer 45 atgtacccat acgacgtccc agactacgcc atggagaaat ttcat 45 46 18 DNA Artificial Sequence Primer 46 accctggttg tggcagta 18 47 17 DNA Artificial Sequence Primer 47 cagcccatga atccatc 17 48 17 DNA Artificial Sequence Primer 48 tgccttcggt ctgaaag 17 49 31 DNA Artificial Sequence Primer 49 aggaaagctt gcccaagaga atagttaatg c 31 50 30 DNA Artificial Sequence Primer 50 aggcgaattc catatcatca atgccaccag 30 51 20 DNA Artificial Sequence Primer 51 tccaccccac tacctgactc 20 52 20 DNA Artificial Sequence Primer 52 cccttctgat gaccacaggt 20 53 9 PRT Artificial Sequence Peptide 53 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 

What is claimed is:
 1. An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, and complements thereof.
 2. A vector comprising the isolated nucleic acid molecule of claim
 1. 3. A host cell comprising the vector of claim
 2. 4. A method of producing an expression product encoded by an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, the method comprising culturing a host cell comprising the isolated nucleic acid molecule under conditions to cause expression of the expression product.
 5. An isolated nucleic acid molecule consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, and complements thereof, wherein the isolated nucleic acid molecule encodes an expression product.
 6. A vector comprising the isolated nucleic acid molecule of claim
 5. 7. A host cell comprising the vector of claim
 6. 8. A method of producing an expression product encoded by an isolated nucleic acid molecule consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, the method comprising culturing a host cell comprising the isolated nucleic acid molecule under conditions to cause expression of the expression product.
 9. A recombinant DNA molecule comprising the nucleic acid molecule of claim 1 or
 5. 